US 20220325332A1 

IN 

( 19 ) United States 

( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2022/0325332 A1 

Balijepalli et al . ( 43 ) Pub . Date : Oct. 13 , 2022 

( 54 ) AGILE NUCLEIC ACID SENSOR AND Publication Classification 

MEASURING A BIOMARKER 

( 71 ) Applicant :Government of the United States of 

America , as represented by the 

Secretary of Commerce , Gaithersburg , MD ( US ) 

( 51 ) Int . Ci . C12Q 1/6825 ( 2006.01 ) 

BOIL 3/00 ( 2006.01 ) 

( 52 ) U.S. CI . CPC C12Q 1/6825 ( 2013.01 ) ; BOIL 3/502715 

( 2013.01 ) 

( 57 ) ABSTRACT 

( 72 ) Inventors : Arvind Kumar Balijepalli , Washington , DC ( US ) ; Jacob Michael 

Majikes , Gaithersburg , MD ( US ) ; 

Alokik Kanwal , Gaithersburg , MD 

( US ) ; Peter Michael Vallone , Potomac , MD ( US ) ; Kevin Michael Kiesler , Rockville , MD ( US ) ; Erica Lee 

Romsos , Ijamsville , MD ( US ) ; 

Anthony José Kearsley , Hanover , MD 

( US ) 

a 

( 21 ) Appl . No .: 17 / 845,682 

An agile nucleic acid sensor includes : a DNA switch ; an analysis substrate in electrostatic communication with the 

DNA switch and that produces a biomarker electrical signal ; 

a transduction member that receives the biomarker electrical 

signal and produces a transduction signal ; a sensor counter 

electrode in electrical communication with and capacitively coupled to the analysis substrate and that receives a counter 

electrode voltage ; a sensor reference electrode in electrical communication and capacitively coupled to the analysis substrate and that produces a feedback signal based on electrical interactions with a composition that is in fluid 

contact with the feedback signal and the analysis substrate ; 

and a voltage follower in electrical communication with the 

sensor counter electrode and the sensor reference electrode 

and that receives the feedback signal from the sensor refer 

ence electrode and produces the counter electrode voltage for the sensor counter electrode based on the feedback signal . 

( 22 ) Filed : Jun . 21 , 2022 

Related U.S. Application Data 

( 63 ) Continuation - in - part of application No. 17 / 360,008 , filed on Jun . 28 , 2021 . 

( 60 ) Provisional application No. 63 / 212,753 , filed on Jun . 21 , 2021 , provisional application No. 63 / 045,366 , filed on Jun . 29 , 2020 . 

200 

108 

205 

209 -101 

109 . 117 -115 

116 

-118 4 + 114 

110 107 

w ! 

113 102 

101 

103 112 
Patent Application Publication Oct. 13 , 9 2022 Sheet 1 of 19 US 2022/0325332 A1 

200 

108 ??????????????????????????????? 

205 

111 209 101 

109 112 115 

116 

118 } 

TO 103 

1141 

110 107 

2 

? : ????????? **** .. : 

113 102 

101 

103 112 

FIG . 1 
Patent Application Publication Oct. 13 , 9 2022 Sheet 2 of 19 US 2022/0325332 A1 

31 200 M 

108 

205 

sistent 101 111 209 

105 109 117 115 

} 116 118 GTO TO103 

119 

www 

********** 115 

} WWWW 114 

110 SA 107 I 

mittit 106 proporcionar igeno 

113 102 

0 

???? 101 

103 112 

FIG . 2 
Patent Application Publication Oct. 13 , 9 2022 Sheet 3 of 19 US 2022/0325332 A1 

( A ) 202 

209 

203 

219 219 

206 

219 219 

210 , 218 + 

201 

WAVAA 219 219 

t 

1 

1 212 | 108 111 211 205 1 1 

1 

101 - 

r 

( B ) 209 

202 201 212 

219 219 

206 210 , 218 

219 219 

203 

219 219 

1 1 I 

1 205 | 108 1 111 1 

? 

101 

211 

FIG . 3 
Patent Application Publication Oct. 13 , 9 2022 Sheet 4 of 19 US 2022/0325332 A1 

( A)( 210 218 

2191 2192 

219.6 

219.5 

219.4 219.3 

( C)210 218 

( B ) 218 210 

219.1 219.1 

219.6 219.2 219.2 

219.3 

Le ? 219.5 219.3 

219.4 

( E ) 210 218 219 

218 219 

( D ) 210 218 219 218 219 

218 Sot 54 

30 42 

219 

218 219 

218 219 

218 219 andao 

66 78 

FIG . 4 
Patent Application Publication Oct. 13 , 9 2022 Sheet 5 of 19 US 2022/0325332 A1 

203 

( ( A ) 

215 216 

weiten 

Aptamer Exchange Region 

( Molecular Adapter ) 

( 8 ) 

209 DNA switch in latched configuration 202 203 

212 206 219 216 

210 , 218 moeten 215 

20 219 

211 205 Analyte & 

Molecular Adapter 

209 DNA switch in unlatched configuration 

212 202 888 

219 

206 

210 , 218 

203 

2019 $

211 205 

FIG . 5 
Patent Application Publication Oct. 13 , 9 2022 Sheet 6 of 19 US 2022/0325332 A1 

501 101 

HULLIT 

Sensor A 

Sensor B 

Sensor a 

502 

FIG . 6 
Patent Application Publication Oct. 13 , 9 2022 Sheet 7 of 19 US 2022/0325332 A1 

501 101 

Sample 

503 

FIG . 7 
Patent Application Publication Oct. 13 , 9 2022 Sheet 8 of 19 US 2022/0325332 A1 

100 nm 

= 

bem com . 20 nm 

} Na 6.6 mm 

FIG . 8 
Patent Application Publication Oct. 13 , 9 2022 Sheet 9 of 19 US 2022/0325332 A1 

Applying an AC electric field to sensor counter electrode108 

Receiving a response current or voltage via working electrode 104 

Estimating an impedance resulting from change in surface properties 

of working electrode 104 due to analyte interactions with DNA nanostrutures 

Repeating impedance measurements to construct a time series of the 

changes in surface charge of working electrode 104 

Extracting the mean kinetic association and dissociation rates from the 

impedance time - series 

Aggregating mean kinetic association and dissociation rates from multilple elements in substrate 101 to construct a fingerprint of interactions 

FIG . 9 
120 Reading kinetic fingerprint fromsensor array

W 

Al -Enhanced Simulator 127

Patent Application Publication 

123 

122 

Updating sensorarray fingereprint training 

data forAlEnhanced -Simulator 

121 

124 and sensor Providing physics modelsdevice 

Simulator Calibrating Almachine learning models

and sesor Providing uncertainties parameter 

Oct. 13 , 2022 Sheet 10 of 19 

125 

Physics enhanced training datawith uncertainty quantihcation 

126 

classification analyte Updating data training 

Classifying analyte typewith machine learning model

classification Analyte 

US 2022/0325332 A1 

FIG .10
Patent Application Publication Oct. 13 , 9 2022 Sheet 11 of 19 US 2022/0325332 A1 

HYV * XXXXX **** 4XX . WWW 

Sensors : DNA Nanotech . Electronics 

• Tunable Specificity 

Measures BOTH 

Binding and Kinetic 

Sensor Validation 

Genetic SRMS 

PCR Verification 

U 

0 OL lo - WWW M WW 

Model Form 

Calibration High - Quality Training Data Measurement Specific 

Al Training 3 

MywamwwwxMWMMWWMWMMMWM 

Outputs 

• Transformative biometrology where 

sensor function is altered by Al . • Implicit uncertainty quantification ( UQ ) . • Comprehensive training data generation . • Rapid and robust Al model development for new applications . 

FIG . 11 
Patent Application Publication Oct. 13 , 9 2022 Sheet 12 of 19 US 2022/0325332 A1 

80 

* 

? 

* 

> 

Cp (nF)? 40 * 

? * 

3 ***************************** V DL . 20 * 

Sá 

1000 104 105 106 

F ( Hz ) 

Cint 

? 

I origami 

F200 HZ 

NR - DL 

FIG . 12 
Patent Application Publication Oct. 13 , 9 2022 Sheet 13 of 19 US 2022/0325332 A1 

K = 8.5 min 

7.34 

7.33 

Cp nF()7.32 

7.31 

7.30 

............................. 

0 10 20 30 40 

t ( min ) 

FIG . 13 
Patent Application Publication Oct. 13 , 9 2022 Sheet 14 of 19 US 2022/0325332 A1 

Analyte 

Antidote 7.21 

7.20 

Cp (nF)7.19 

7.18 

7.17 

........................................... 

0 10 20 30 40 50 

t ( min ) 

FIG . 14 
Patent Application Publication Oct. 13 , 9 2022 Sheet 15 of 19 US 2022/0325332 A1 

Alfine Transform 

FIG . 15 
Patent Application Publication Oct. 13 , 9 2022 Sheet 16 of 19 US 2022/0325332 A1 

Viral particles DNA / RNA 

100 copies ) Extraction 

Al - based Recognition 

::: Robust UQ 

::: Repurpose Sensors 

Modular and Tunable Highly Multiplexed 

DNA - Electronic Array 

NO PCR 

enrichment 

Days 

Potential 

new strain 

Updated 

tests 

FIG . 16 
Patent Application Publication Oct. 13 , 9 2022 Sheet 17 of 19 US 2022/0325332 A1 

Viral particles DNA / RNA 

100 copies ) Extraction 

Surveillance Testing 

www 

Sequencing Assay Development ARCH o microuran Rapid tests 

POR ws 

enrichment Weeks 

Updated 

FIG . 17 
Patent Application Publication Oct. 13 , 9 2022 Sheet 18 of 19 US 2022/0325332 A1 

analyte on s 

analyte off 

Sensor array analyte 

( 10 molecules ) 

analyte 

LLLLLL signal time 

mutant 

JUUL time 

FIG . 18 
Patent Application Publication Oct. 13 , 9 2022 Sheet 19 of 19 US 2022/0325332 A1 

S s 

SIST 

mutant 

> 10s molecules ; not detected ) 

analyte ( > 10 % molecules ) 

signal 

concentration 

FIG . 19 
US 2022/0325332 Al Oct. 13 , 2022 

1 

AGILE NUCLEIC ACID SENSOR AND 

MEASURING A BIOMARKER 

CROSS REFERENCE TO RELATED 

APPLICATIONS 

[ 0001 ] This application is a continuation - in - part and 

claims benefit of the following applications : U.S. Provi 

sional Patent Application Ser . No. 63 / 212,753 , filed Jun . 21 , 2021 ; and U.S. patent application . Ser . No. 17 / 360,008 , filed 

Jun . 28 , 2021 , which claims benefit to Provisional Patent Application Ser . No. 63 / 045,366 , filed Jun . 29 , 2020 , all of which are incorporated by reference in their entirety . 

trode voltage by the voltage follower ; subjecting the sensor 

counter electrode to the counter electrode voltage from the 

voltage follower ; contacting the DNA switch with a chemi 

cal analyte comprising the biomarker ; producing , by the 

analysis substrate , a biomarker electrical signal in response to the chemical analyte contacting the DNA switch ; receiv 

ing , by the transduction member , the biomarker electrical signal from the analysis substrate and producing a transduc 

tion signal from the biomarker electrical signal ; determining the impedance of the sensor working electrode from the transduction signal ; estimating kinetic rate constants for 

each contact between the chemical analyte and the DNA 

switch ; and combining the kinetic rate constants and pro ducing a mean kinetic rate constant with uncertainty quan tification for the kinetic rate constants ; aggregating the mean 

kinetic rate constants for the plurality of agile nucleic acid 

sensors in the agile nucleic acid sensor array ; and producing 

a kinetic fingerprint for the chemical analyte from the mean 

kinetic rate constants . 

STATEMENT REGARDING FEDERALLY 

SPONSORED RESEARCH 

[ 0002 ] This invention was made with United States Gov 

ernment support from the National Institute of Standards and Technology ( NIST ) , an agency of the United States Depart 

ment of Commerce . The Government has certain rights in 

this invention . BRIEF DESCRIPTION OF THE DRAWINGS 

BRIEF DESCRIPTION 

[ 0003 ] Disclosed is an agile nucleic acid sensor for mea suring a biomarker , the agile nucleic acid sensor comprising : 

a DNA switch that contacts and selectively duplexes with a 

chemical analyte comprising the biomarker ; an analysis substrate in electrostatic communication with the DNA 

switch that is disposed on the analysis substrate , such that 

when the chemical analyte duplexes with the DNA switch , the analysis substrate produces a biomarker electrical signal ; 

a transduction member in electrical communication with the 

analysis substrate and that receives the biomarker electrical signal and produces a transduction signal in response to 

receiving the biomarker electrical signal ; a sensor counter electrode in electrical communication with and capacitively coupled to the analysis substrate and that receives a counter 

electrode voltage ; a sensor reference electrode in electrical communication and capacitively coupled to the analysis substrate and that produces a feedback signal based on electrical interactions with a composition that is in fluid 

contact with the feedback signal and the analysis substrate ; 

and a voltage follower in electrical communication with the 

sensor counter electrode and the sensor reference electrode 

and that receives the feedback signal from the sensor refer 

ence electrode and produces the counter electrode voltage for the sensor counter electrode based on the feedback 

signal . [ 0004 ] Disclosed is a process for measuring a biomarker 

with an agile nucleic acid sensor array , the process com prising : operating the agile nucleic acid sensor array that 

comprises : a plurality of agile nucleic acid sensors arranged in array and that individually comprise : a DNA switch ; an analysis substrate in electrostatic communication with the 

DNA switch that is disposed on the analysis substrate ; a 

transduction member in electrical communication with the 

analysis substrate ; a sensor counter electrode in electrical communication with and capacitively coupled to the analy 

sis substrate ; a sensor reference electrode in electrical com munication and capacitively coupled to the analysis sub 

strate ; and a voltage follower in electrical communication 

with the sensor counter electrode and the sensor reference 

electrode ; for individual agile nucleic acid sensors in the 

agile nucleic acid sensor array : producing a counter elec 

[ 0005 ] The patent or application file contains at least one drawing executed in color . Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee . [ 0006 ] The following description cannot be considered limiting in any way . Various objectives , features , and advan 

tages of the disclosed subject matter can be more fully appreciated with reference to the following detailed descrip tion of the disclosed subject matter when considered in 

connection with the following drawings , in which like 

reference numerals identify like elements . [ 0007 ] FIG . 1 shows an agile nucleic acid sensor , accord 

ing to some embodiments . [ 0008 ] FIG . 2 shows an agile nucleic acid sensor , accord 

ing to some embodiments . [ 0009 ] FIG . 3 shows a DNA switch before binding a 

chemical analyte in panel A and after binding the chemical 

analyte in panel B , wherein the DNA switch is configured for robust signal amplification upon analyte binding , accord 

ing to some embodiments . [ 0010 ] FIG . 4 shows , according to some embodiments : 

panel A ) a perspective view of a nucleic acid core that 

includes a DNA helix bundle for six DNA helixes ; ( panel B ) 

a side view of the nucleic acid core shown in panel A ; ( panel 

C ) a cross - section along line A - A of the nucleic acid core 

shown in panel B ; ( panel D ) a cross - section of a DNA helix 

bundle that includes ten DNA helixes arranged in a lemnis 

cate configuration ; and ( panel E ) cross - sections of various 

DNA helix bundles that includes different numbers of DNA 

helixes indicated by the number located centrally for each 

DNA helix bundle . 

[ 0011 ] FIG . 5 shows , according to some embodiments : 

( panel A ) formation of a chemical analyte from an aptamer and an analyte ; ( panel B ) a DNA switch in a latched 

configuration wherein a second helix strand is hybridized to 

a first helix strand ; and ( panel C ) the DNA switch in an 

unlatched configuration wherein the second helix strand is unhybridized to the first helix strand that is hybridized to the chemical analyte , wherein molecular adapters ( e.g. , aptam 

ers ) conjugate to probe DNA molecules that provide a 

variety of recognition elements . 

a 

a 
US 2022/0325332 A1 Oct. 13 , 2022 

2 

[ 0012 ] FIG . 6 shows an arrangement for disposing DNA 

switches on analysis substrates , according to some embodi 

ments . 

[ 0013 ] FIG . 7 shows an arrangement for introducing chemical analytes to DNA switches disposed on analysis substrates , according to some embodiments . [ 0014 ] FIG . 8 shows a notched rectangle origami for 

sensing a biomarker , according to some embodiments . [ 0015 ] FIG . 9 shows steps in measuring a biomarker with 

an agile nucleic acid sensor , according to some embodi 

ments . 

[ 0016 ] FIG . 10 shows steps in measuring a biomarker with 

an agile nucleic acid sensor , according to some embodi 

ments . 

[ 0017 ] FIG . 11 shows steps in measuring a biomarker with 

an agile nucleic acid sensor , according to some embodi 

ments . 

[ 0018 ] FIG . 12 shows several graphs for quantifying sur 

face functionalization of sensor working electrode with notched rectangle origami , according to some embodiments . [ 0019 ] FIG . 13 shows a kinetic response of notched rect angle origami to analyte , according to some embodiments . [ 0020 ] FIG . 14 shows a kinetic recovery of notched rect angle origami demonstrating reversibility , according to 

some embodiments . [ 0021 ] FIG . 15 shows qPCR data correction with an affine 

transform , according to some embodiments . [ 0022 ] FIG . 16 shows a measurement outline for measur ing a biomarker , according to some embodiments . [ 0023 ] FIG . 17 shows a conventional measurement out line , according to a comparative example . [ 0024 ] FIG . 18 shows data collection from an agile nucleic 

acid sensor , according to some embodiments . [ 0025 ] FIG . 19 shows conventional data collection , according to a comparative example . 

chemical analytes . High - dimensional data including the 

unique molecular signatures are involved in decoding by AI 

methods and quantifying measurement errors and uncer tainty ( UQ ) . The agile nucleic acid sensor array is a repro grammable label - free and PCR - free biomarker detection 

chip that can be used in many areas such as medical 

diagnostics and surveillance of public health threats . 

[ 0029 ] Agile nucleic acid sensor 200 measures a bio 

marker . In an embodiment , with reference to FIG . 1 , FIG . 2 , FIG . 3 , FIG . 4 , FIG . 5 , FIG . 6 , FIG . 7 , FIG . 8 , FIG . 9 , FIG . 10 , and FIG . 11 , agile nucleic acid sensor 200 includes : a 

DNA switch 209 that contacts and selectively duplexes with 

a chemical analyte 203 including the biomarker ; an analysis 

substrate 205 in electrostatic communication with the DNA 

switch 209 that is disposed on the analysis substrate 205 , such that when the chemical analyte 203 duplexes with the 

DNA switch 209 , the analysis substrate 205 produces a 

biomarker electrical signal 115 ; a transduction member 114 

in electrical communication with the analysis substrate 205 

and that receives the biomarker electrical signal 115 and 

produces a transduction signal 116 in response to receiving the biomarker electrical signal 115 ; a sensor counter elec 

trode 108 in electrical communication with and capacitively coupled to the analysis substrate 205 and that receives a 

counter electrode voltage 117 ; a sensor reference electrode 111 in electrical communication and capacitively coupled to 

the analysis substrate 205 and that produces a feedback 

signal 118 based on electrical interactions with a composi tion that is in fluid contact with the feedback signal 118 and 

the analysis substrate 205 ; and a voltage follower 109 in 

electrical communication with the sensor counter electrode 

108 and the sensor reference electrode 111 and that receives 

the feedback signal 118 from the sensor reference electrode 

111 and produces the counter electrode voltage 117 for the 

sensor counter electrode 108 based on the feedback signal 118 . 

[ 0030 ] In an embodiment , with reference to FIG . 3 , FIG . 4 , and FIG . 5 , DNA switch 209 includes : a DNA nanostruc 

ture framework 202 disposed on the analysis substrate 205 

and including a nucleic acid core 210 , a first helix strand 211 

protruding from the nucleic acid core 210 and attached to the 

analysis substrate 205 , and a second helix strand 212 pro truding from the nucleic acid core 210 such that the second 

helix strand 212 is hybridized to the first helix strand 211 in 

an absence of a chemical analyte 203 that preferentially hybridizes to the first helix strand 211 as compared with the 

second helix strand 212 , and the second helix strand 212 

dissociates from the first helix strand 211 when the first helix 

strand 211 is in a presence of the chemical analyte 203 ; a 

particle strand 206 hybridized to the second helix strand 

212 ; and a reporter particle 201 attached to the particle strand 206 and disposed proximate to the analysis substrate 

205 when the second helix strand 212 is hybridized to the 

first helix strand 211 in absence of the chemical analyte 203 

and that changes the electrical potential of the analysis substrate 205 depending on whether the second helix strand 

212 is hybridized to the first helix strand 211 . 

[ 0031 ] In an embodiment , when the chemical analyte 203 

is hybridized to the first helix strand 211 , the reporter particle 201 remains attached to the nucleic acid core 210 , and is sterically or thermodynamically precluded from inter 

acting with first helix strand 211 . 

DETAILED DESCRIPTION 

a 

2 

[ 0026 ] A detailed description of one or more embodiments is presented herein by way of exemplification and not 

limitation . 

[ 0027 ] Nucleic acids , e.g. , DNA and RNA are building blocks for life . Mutations in nucleic acids from inherited or 

environmental factors can result in diseases such as cancers . Viruses employ rapid mutations to hijack cells and evade the 

immune system . Surveillance of nucleic acids can be used to 

monitor public health . Mutations in biomarkers can outpace development or deployment of high - specificity diagnostic 

tests . There is a need for a novel and agile biomarker 

metrology , as evidenced by pandemics such as attending spread of SARS COV - 2 mutants that challenged conven tional diagnostic testing infrastructure . The agile nucleic 

acid sensor and process for measuring a biomarker disclosed herein meet this need and provide a biomarker metrology that combines DNA nanostructure - enhanced detectors with artificial intelligence ( AI ) frameworks with flexibility to 

allow rapid reprogramming in the field . [ 0028 ] The agile nucleic acid sensor and process for measuring a biomarker have high sensitivity in an absence 

of polymerase chain reaction ( PCR ) . PCR is a sample enrichment technique with disadvantages that include a significant wait for analysis results and system complexity . The agile nucleic acid sensor array disclosed herein involves 

DNA nanotechnology to make arrays of electronic sensors 

that produce unique molecular signatures for different 
US 2022/0325332 A1 Oct. 13 , 2022 

3 

a 

[ 0032 ] In an embodiment , the transduction member 114 includes a transimpedance amplifier 107. In some embodi 

ment , the transduction member 114 further includes a tran 

sistor 105 . 

[ 0033 ] In an embodiment ,agile nucleic acid sensor 200 

includes a drain voltage source 106 in electrical communi 

cation with the transistor 105 and that produces a drain 

voltage 119 for the transistor 105 . [ 0034 ] In an embodiment , agile nucleic acid sensor 200 

includes voltage setpoint source 110 in electrical commu nication with the voltage follower 109 and that produces the 

counter electrode voltage 117 for the voltage follower 109 . [ 0035 ] In an embodiment ,agile nucleic acid sensor 200 

includes a signal processor 103 in electrical communication 

with the transduction member 114 and that receives the 

transduction signal 116 from the transduction member 114 . [ 0036 ] In an embodiment ,agile nucleic acid sensor 200 

includes an interface pad 112 in electrical communication the transimpedance amplifier 107 . [ 0037 ] In an embodiment ,agile nucleic acid sensor 200 

includes a substrate 101 on which the DNA switch 209 , the 

analysis substrate 205 , the transduction member 114 , the 

sensor counter electrode 108 , the sensor reference electrode 

111 , and the voltage follower 109 are disposed . [ 0038 ] In an embodiment ,agile nucleic acid sensor 200 

includes a plurality of agile nucleic acid sensors 200 arranged in array . In an embodiment , agile nucleic acid 

sensor 200 includes a signal processor 103 , wherein , for 

each agile nucleic acid sensor 200 , the signal processor 103 

is in electrical communication with a transduction member 

114 , and the signal processor 103 receives a transduction 

signal 116 from the transduction member 114. In an embodi 

ment , agile nucleic acid sensor 200 includes a plurality of interface pads 112 , such that each transimpedance amplifier 107 is in electrical communication with an interface pad . In 

an embodiment , agile nucleic acid sensor 200 includes a 

substrate 101 on which the agile nucleic acid sensors 200 are 

disposed . In an embodiment , the agile nucleic acid sensors 

200 individually include : a DNA switch 209 that contacts 

and selectively duplexes with a chemical analyte 203 com 

prising the biomarker ; an analysis substrate 205 in electro 

static communication with the DNA switch 209 that is 

disposed on the analysis substrate 205 , such that when the 

chemical analyte 203 duplexes with the DNA switch 209 , the analysis substrate 205 produces a biomarker electrical 

signal 115 ; a transduction member 114 in electrical com 

munication with the analysis substrate 205 and that receives 

the biomarker electrical signal 115 and produces a transduc tion signal 116 in response to receiving the biomarker 

electrical signal 115 ; a sensor counter electrode 108 in 

electrical communication with and capacitively coupled to 

the analysis substrate 205 and that receives a counter elec 

trode voltage 117 ; a sensor reference electrode 111 in 

electrical communication and capacitively coupled to the 

analysis substrate 205 and that produces a feedback signal 118 based on electrical interactions with a composition that 

is in fluid contact with the feedback signal 118 and the analysis substrate 205 ; and a voltage follower 109 in elec 

trical communication with the sensor counter electrode 108 

and the sensor reference electrode 111 and that receives the 

feedback signal 118 from the sensor reference electrode 111 

and produces the counter electrode voltage 117 for the 

sensor counter electrode 108 based on the feedback signal 118 . 

[ 0039 ] In an embodiment , the DNA switch 209 includes : 

a DNA nanostructure framework 202 disposed on the analy sis substrate 205 and including a nucleic acid core 210 , a first 

helix strand 211 protruding from the nucleic acid core 210 

and attached to the analysis substrate 205 , and a second helix 

strand 212 protruding from the nucleic acid core 210 such 

that the second helix strand 212 is hybridized to the first 

helix strand 211 in an absence of a chemical analyte 203 that 

preferentially hybridizes to the first helix strand 211 as 

compared with the second helix strand 212 , and the second 

helix strand 212 dissociates from the first helix strand 211 

when the first helix strand 211 is in a presence of the 

chemical analyte 203 ; a particle strand 206 hybridized to the 

second helix strand 212 ; and a reporter particle 201 attached 

to the particle strand 206 and disposed proximate to the 

analysis substrate 205 when the second helix strand 212 is 

hybridized to the first helix strand 211 in absence of the chemical analyte 203 and that changes the electrical poten tial of the analysis substrate 205 depending on whether the 

second helix strand 212 is hybridized to the first helix strand 

211. In an embodiment ,when the chemical analyte 203 is hybridized to the first helix strand 211 : the reporter particle 201 remains attached to the nucleic acid core 210 , and is sterically or thermodynamically precluded from interacting 

with first helix strand 211 . 

[ 0040 ] Various molecular structures described herein 

involve nucleic acids . As used herein , a “ nucleic acid ” can 

include deoxyribonucleic acid ( DNA ) , ribonucleic acid 

( RNA ) , or artificial nucleic acids , such as a peptide nucleic 

acid ( PNA ) . The molecular structure may include one type of nucleic acid ( e.g. , DNA ) , or more than one type in some 

cases , which may form part of the same molecule or different 

molecules assembled together in a supramolecular assembly defining the overall molecular structure . Typically , the 

nucleic acid is a polymeric molecule including one or more 

" bases ” ( usually nitrogenous ) connected to a backbone 

structure , which can be a sugar - phosphate backbone ( e.g. , as 

in DNA or RNA ) or a peptide backbone ( e.g. , as in PNA ) . [ 0041 ] The sugars within the nucleic acid , when present , may be , for example , ribose sugars ( as in RNA ) , or deoxy ribose sugars ( as in DNA ) . In some cases , the nucleic acid 

can include ribose and deoxyribose sugars . Examples of 

bases that may be found within a nucleic acid include , but 

are not limited to , the naturally - occurring bases ( e.g. , adenosine or “ A , ” thymidine or “ T , ” guanosine or “ G , " 

cytidine or “ C ” or uridine or “ U ” ) . The bases typically interact on a specific basis ( i.e. , guanosine interacts with cytidine via hydrogen bonding and vice versa , and adenos 

ine interacts with thymidine or uridine via hydrogen bonding and vice versa ) . In some cases , the nucleic acid may include 

nucleoside analogs ( e.g. , 2 - aminoadenosine , 2 - thiothymi dine , inosine , pyrrolopyrimidine , 3 - methyladenosine , C5 - bromouridine , C5 - fluorouridine , C5 - iodouridine , C5 - propynyluridine , C5 - propynylcytidine , C5 - methylcyti dine , 7 - deazaadenosine , 7 - deazaguanosine , 8 - oxoadenosine , 8 - oxoguanosine , 06 - methylguanosine , 2 - thiocytidine , 2 - aminopurine , 2 - amino - 6 - chloropurine , 2,6 - diaminopu rine , hypoxanthine ) , chemically or biologically modified 

bases ( e.g. , methylated bases ) , intercalated bases , modified 

sugars ( 2 ' - fluororibose , arabinose , or hexose ) , modified phosphate moieties ( e.g. , phosphorothioates or 5 ' - N - phos phoramidite linkages ) , or other naturally and non - naturally occurring bases substitutable into the nucleic acid , including 

a 
US 2022/0325332 A1 Oct. 13 , 2022 

4 

a 

a 

substituted and unsubstituted aromatic moieties . Other suit 

able base or backbone modifications can occur . [ 0042 ] The nucleic acid can be single - stranded or double 

stranded , i.e. , formed of two strands ( or of the same strand 

looped back on itself , such as in a hairpin turn or a stem - loop structure ) associated with each other via hydrogen bonding , e.g. , via guanosine / cytidine base - pair interactions , adenos 

ine / thymidine base - pair interactions , adenosine / uridine 

base - pair interactions , etc. 

[ 0043 ] The nucleic acids can be present within a molecular 

structure as a bundle , which can include two or more 

non - complementary nucleic acid portions associated with 

each other . The nucleic acids forming the bundles can be single stranded or double stranded , and the non - complemen tary nucleic acid portions can be part of the same nucleic 

acid molecule or part of different nucleic acid molecules . For 

instance , there may be 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 14 , 16 , 18 , 20 , 24 , 30 , 42 , 54 , 66 , 78 , 90 , or more non - complementary nucleic acid portions associated with each other as part of a 

bundle . There can be other nucleic acid strands associated 

with one or more portions of the nucleic acids forming the nucleic acid bundle , e.g. , to provide stability . [ 0044 ] It should be noted that , in a bundle of nucleic acid , not all of the nucleic acid strands need run from one end of 

the bundle to the other . For example , one or more nucleic 

acid strands may run from a first end of the bundle , through 

a hairpin turn or a stem - loop structure , back to the first end 

of the bundle ( or may go through more than one hairpin turn 

or a stem - loop structure , in some cases ) ; or a nucleic acid 

strand may end within the bundle . [ 0045 ] In some cases , the bundles can define a nanotube . The nanotube can have a hollow center , with nucleic acid 

strands arranged around the center ( thus , a double strand of 

DNA , by itself , is not a nanotube , as the two sugar - phos phate backbones forming the DNA are interconnected by bases hydrogen bonded to each other , which thus does not 

result in a hollow center ) . The nanotube may be circular or elliptical , or in some cases , the nanotube may have polygo nal shapes such as a hexagon . In some cases , the nanotube 

may have more than one hollow center , e.g. , having the 

shape of a lemniscate . Non - limiting examples of such nano 

tubes are shown in FIG . 3A ( perspective view of a six - helix 

nucleic acid bundle ) , FIG . 3B ( side view of the six - helix 

nucleic acid bundle shown in panel A ) , FIG . 3C ( cross 

section along line A - A of the six - helix nucleic acid bundle 

shown in panel B ) , FIG . 3D ( a ten - helix nucleic acid bundle , having a lemniscate shape with two hollow centers ; thus , more than one hollow center may be present within the 

nanotube ) , and FIG . 3E ( bundles with the number of nucleic 

acid strands present within the nanotube shown in the center 

of each nanotube ) . The nucleic acid portions forming the 

bundled nanotube may be part of the same nucleic acid 

molecule or may be part of different nucleic acid molecules . In some cases , the nanotube may be formed from an even 

number of nucleic acid strands ( e.g. , 4 , 6 , 8 , 10 , 12 , etc. ) . In 

certain embodiments , other molecules may be present within 

the nanotube , for example , to provide stability to the nano 

tube structure . 

[ 0046 ] In some embodiments , one or more of the nucleic 

acid bundles or nanotubes within the molecular structure 

may be fabricated from one or more relatively long nucleic acids , e.g. , having lengths of at least about 500 nucleotides , at least about 1,000 nucleotides , at least about 3,000 nucleo 

tides , at least about 10,000 nucleotides , at least about 30,000 

nucleotides , etc. Such a nucleic acid may be referred to as a 

nucleic acid scaffold . The nucleic acid scaffold may form a 

single bundle or nanotube , or may include different parts of 

different bundles or nanotubes in the final molecular struc 

ture . For instance , a nucleic acid scaffold may wrap in 

various ways around the molecular structure , e.g. , forming various nucleic acid bundles or nanotubes defining the 

molecular structure . In some cases , a nucleic acid may form 

a first portion of a nucleic acid bundle and a second portion of the same nucleic acid bundle ( or a different one ) , where 

the first and second portions forming the nucleic acid bundle 

are not complementary . In one set of embodiments , the 

nucleic acid scaffolds are substantially free of self - comple mentary regions and / or repeat units , as discussed below . In 

certain embodiments of the invention , the nucleic acid 

scaffolds are immobilized to form one or more bundles or 

nanotubes , and ultimately a three - dimensional structure , using one or more nucleic acid stabilizers able to associate 

with two or more portions of the nucleic acid . In certain 

embodiments , the structures can have other shapes , e.g. , notched rectangles , as well as other planar or three - dimen 

sional structures . 

[ 0047 ] One source of a nucleic acid having such charac teristics is bacteriophage DNA , for example , M13 bacterio 

phage . The DNA in such bacteriophages may be single stranded DNA , and have substantially few self - complemen tary regions ( e.g. , only 2 hairpin regions may form ) , and a 

length of about 7,000 nucleotides . The DNA can be removed 

from the bacteriophage using DNA isolation techniques known to those of ordinary skill in the art , for example , by using lysis buffer ( e.g. , comprising an alkaline environment 

or surfactant ) followed by centrifugation at greater than 

10,000 RCF ( relative centrifugal force ) to separate the DNA . [ 0048 ] The molecular structure may be stabilized , in some 

cases , by nucleic acid stabilizers able to associate with two 

or more nucleic acid portions . For example , a nucleic acid stabilizer may include a first portion complementary to a 

first nucleic acid strand ( e.g. , a nucleic acid scaffold ) and a second portion complementary to a second nucleic acid 

strand . The first and second portions may be part of the same 

nucleic acid molecule , or may be part of different molecules . In some cases , the nucleic acid stabilizer may be formed 

essentially from nucleic acid . A nucleic acid stabilizer may have a length of between about 20 nucleotides and about 100 

nucleotides , for example , between about 35 nucleotides and about 45 nucleotides , or about 40 nucleotides . As the first portion of the nucleic acid stabilizer binds to the first nucleic 

acid portion and the second portion binds to the second 

nucleic acid portions , the two portions are substantially immobilized , relative to each other , due to the presence of 

the nucleic acid stabilizer . Thus , the two portions are not 

able to move apart , or at least are not able to move far apart , and remain associated together . By using a plurality of 

nucleic acid stabilizers , e.g. , targeted to different nucleic 

acids or different portions of nucleic acids , one or more nucleic acids can be stabilized in a substantially rigid configuration , e.g. , as a bundle or a nanotube . In addition , these can further be configured as part of larger molecular 

structures . A technique for forming nucleic acid stabilizers is 

found in Rothemund , P. W. K. , “ Folding DNA to Create 

Nanoscale Shapes and Patterns , ” Nature , 440 : 297-302 

( 2006 ) , which is incorporated by reference in its entirety . [ 0049 ] As used herein , " microRNA ” or " miRNA ” 

describes small , non - coding RNA molecules , generally 
US 2022/0325332 A1 Oct. 13 , 2022 

5 

a 

a 

about 15 to about 50 nucleotides in length , specifically 17-23 

nucleotides , that can play a role in regulating gene expres sion through , e.g. , a process referred to as RNA interference 

( RNA ) . RNAi describes a phenomenon whereby the pres 

ence of an RNA sequence that is complementary or anti 

sense to a sequence in a target gene messenger RNA 

( mRNA ) results in inhibition of expression of the target 

gene . Here , miRNAs are processed from hairpin precursors of about 70 or more nucleotides ( pre - miRNA ) that are derived from primary transcripts ( pri - miRNA ) through sequential cleavage by RNAse III enzymes . Moreover , miR 

Base is a comprehensive microRNA database located at www.mirbase.org , incorporated by reference herein in its 

entirety for all purposes . [ 0050 ] The RNAi can be transcribed as an RNA precursor having a hairpin - like structure , cleaved by a dsRNA - cleav ing enzyme having RNase III cleavage activity , and inte 

grated into a protein complex called RISC and can be 

involved in the suppression of translation of mRNA . The 

term “ miRNA ” includes not only a “ miRNA ” represented by 

a particular nucleotide sequence but a “ miRNA ” including a 

precursor of the “ miRNA ” ( pre - miRNA or pri - miRNA ) and having biological functions equivalent to miRNAs encoded 

by these , e.g. , a “ miRNA " encoding a congener ( i.e. , a 

homolog or an ortholog ) , a variant such as a genetic poly morph , and a derivative . Such a “ miRNA " encoding a 

precursor , a congener , a variant , or a derivative can be 

specifically identified using miRBase Release 22 ( http : // www.mirbase.org ) , and examples thereof can include a 

" miRNA ” having a nucleotide sequence hybridizing to a 

complementary nucleotide sequence . It is contemplated that 

miRNA can be a gene product of a miR gene . Such a gene product includes a mature miRNA ( e.g. , a 15- to 25 - nucleo 

tide or 19- to 25 - nucleotide non - coding RNA involved in the suppression of translation of mRNA as described above ) or 

a miRNA precursor ( e.g. , pre - miRNA or pri - miRNA as 

described above ) . [ 0051 ] Complementary polynucleotide and similar refer 

ents such as complementary strand or reverse strand includes a polynucleotidee in a complementary relationship based on A : T ( U ) and G : C base pairs with the full - length sequence of a polynucleotide or a nucleotide sequence derived from the nucleotide sequence by the replacement of 

U with T , or a partial sequence thereof ( here , this full - length 

or partial sequence is referred to as a plus strand for the sake 

of convenience ) . However , such a complementary strand is 

not limited to a sequence completely complementary to the 

nucleotide sequence of the target plus strand and may have 

a complementary relationship to an extent that permits hybridization under stringent conditions to the target plus strand . 

[ 0052 ] The term “ Tm value ” refers to a temperature at which the double - stranded moiety of a polynucleotide is 

denatured into single strands so that the double strands and 

the single strands exist at a ratio of 1 : 1 . [ 0053 ] The term “ aptamer ” indicates oligonucleic acid or 

peptide molecules that are capable to bind a specific target . It is contemplated that the aptamer can include single stranded oligonucleotides and chemically synthesized pep tides that have been engineered through repeated rounds of 

in vitro selection , or equivalent techniques identifiable by a 

skilled person , to bind to various targets . [ 0054 ] As used herein , the term “ nucleotide ” refers to a 

molecule that includes a sugar and at least one phosphate 

group , and optionally also includes a nucleobase . A nucleo 

tide that lacks a nucleobase can be referred to as “ abasic . ” Nucleotides include deoxyribonucleotides , modified deoxy ribonucleotides , ribonucleotides , modified ribonucleotides , peptide nucleotides , modified peptide nucleotides , modified phosphate sugar backbone nucleotides , and mixtures 

thereof . Examples of nucleotides include adenosine mono phosphate ( AMP ) , adenosine diphosphate ( ADP ) , adenosine triphosphate ( ATP ) , thymidine monophosphate ( TMP ) , thy midine diphosphate ( TDP ) , thymidine triphosphate ( TTP ) , cytidine monophosphate ( CMP ) , cytidine diphosphate ( CDP ) , cytidine triphosphate ( CTP ) , guanosine monophos phate ( GMP ) , guanosine diphosphate ( GDP ) , guanosine triphosphate ( GTP ) , uridine monophosphate ( UMP ) , uridine 

diphosphate ( UDP ) , uridine triphosphate ( UTP ) , deoxyade nosine monophosphate ( AMP ) , deoxyadenosine diphos phate ( dADP ) , deoxyadenosine triphosphate ( dATP ) , deoxy thymidine monophosphate ( dTMP ) , deoxythymidine diphosphate ( dTDP ) , deoxythymidine triphosphate ( dTTP ) , deoxycytidine diphosphate ( dCDP ) , deoxycytidine triphos phate ( CTP ) , deoxyguanosine monophosphate ( GMP ) , deoxyguanosine diphosphate ( DGDP ) , deoxyguanosine tri 

phosphate ( dGTP ) , deoxyuridine monophosphate ( dUMP ) , deoxyuridine diphosphate ( dUDP ) , and deoxyuridine tri 

phosphate ( DUTP ) . [ 0055 ] The term “ nucleotide ” also include any “ nucleotide 

analogue which is a type of nucleotide that includes a modified nucleobase , sugar or phosphate moiety compared to naturally occurring nucleotides . Exemplary modified 

nucleobases that can be included in a polynucleotide , whether having a native backbone or analogue structure , include , inosine , xathanine , hypoxathanine , isocytosine , isoguanine , 2 - aminopurine , 5 - methylcytosine , 5 - hydroxym ethyl cytosine , 2 - aminoadenine , 6 - methyl adenine , 6 - methyl guanine , 2 - propyl guanine , 2 - propyl adenine , 2 - thiouracil , 2 - thiothymine , 2 - thiocytosine , 15 - halouracil , 15 - halocyto sine , 5 - propynyl uracil , 5 - propynyl cytosine , 6 - azo uracil , 6 - azo cytosine , 6 - azo thymine , 5 - uracil , 4 - thiouracil , 8 - halo 

adenine or guanine , 8 - amino adenine or guanine , 8 - thiol 

adenine or guanine , 8 - thioalkyl adenine or guanine , 8 - hy droxyl adenine or guanine , 5 - halo substituted uracil or cytosine , 7 - methylguanine , 7 - methyladenine , 8 - azaguanine , 8 - azaadenine , 7 - deazaguanine , 7 - deazaadenine , 3 - deazagua nine , 3 - deazaadenine or the like . As is known in the art , certain nucleotide analogues cannot become incorporated into a polynucleotide , for example , nucleotide analogues such as adenosine 5 ' - phosphosulfate . [ 0056 ] As used herein , the term “ polynucleotide ” refers to 

a molecule that includes a sequence of nucleotides that are 

bonded to one another . Examples of polynucleotides include 

deoxyribonucleic acid ( DNA ) , ribonucleic acid ( RNA ) , and 

analogues thereof .A polynucleotide can be a single stranded 

sequence of nucleotides , such as RNA or single stranded 

DNA , a double stranded sequence of nucleotides , such as 

double stranded DNA , or can include a mixture of a single stranded and double stranded sequences of nucleotides . Double stranded DNA ( dsDNA ) includes genomic DNA , and PCR and amplification products . Single stranded DNA 

( ssDNA ) can be converted to dsDNA and vice - versa . The precise sequence of nucleotides in a polynucleotide can be known or unknown . The following are exemplary examples of polynucleotides : a gene or gene fragment ( for example , a 

probe , primer , expressed sequence tag ( EST ) or serial analy sis of gene expression ( SAGE ) tag ) , genomic DNA , 

? 
US 2022/0325332 A1 Oct. 13 , 2022 

6 

genomic DNA fragment , exon , intron , messenger RNA 

( mRNA ) , transfer RNA , ribosomal RNA , ribozyme , cDNA , recombinant polynucleotide , synthetic polynucleotide , branched polynucleotide , plasmid , vector , isolated DNA of 

any sequence , isolated RNA of any sequence , nucleic acid probe , primer or amplified copy of any of the foregoing . [ 0057 ] As used herein , “ hybridize ” refers to noncovalently binding a first polynucleotide to a second polynucleotide . The strength of the binding between the first and second polynucleotides increases with the complementarity between those polynucleotides . [ 0058 ] As used herein , the term “ protein ” refers to a 

molecule that includes a polypeptide that is folded into a three - dimensional structure . The polypeptide includes moi 

eties that , when folded into the three - dimensional structure , impart the protein with biological activity . [ 0059 ] The term “ sensor ” indicates a device that measures 

a physical quantity and converts it into a signal which can be 

read by an observer or by an instrument .The sensors can be 

calibrated against a known standard . [ 0060 ] The term " detect ” or “ detection ” indicates deter mination of the existence or presence of a chemical analyte 

or other target or signal in a limited portion of space , including but not limited to a sample , a reaction mixture , a 

molecular complex and a substrate including a platform and 

an array . Detection is “ quantitative ” when it refers , relates 

to , or involves the measurement of quantity or amount of chemical analyte or signal ( also referred as quantification ) , which includes but is not limited to any analysis designed to 

determine the amounts or proportions of the chemical ana 

lyte or signal . Detection is " qualitative ” when it refers , relates to , or involves identification of a quality or kind of 

the chemical analyte or signal in terms of relative abundance 

to another chemical analyte or signal , which is not quanti fied . An “ optical detection ” indicates detection performed through visually detectable signals : spectra or images from 

a chemical analyte or a probe attached to the chemical 

analyte . An “ electrical detection ” indicates detection per formed through electrically detectable signals : voltage , elec 

trical current , induction , or capacitance from a chemical analyte or a probe attached to the chemical analyte . [ 0061 ] The term “ chemical analyte ” refers to a substance , compound , or component whose presence or absence in a 

sample is detected through hybridization . Chemical analytes include biomolecules and in particular biomarkers . The term 

“ biomolecule ” indicates a substance compound or compo nent associated to a biological environment including but 

not limited to sugars , amino acids , peptides proteins , oligo nucleotides , polynucleotides , polypeptides , organic mol 

ecules , haptens , epitopes , biological cells , parts of biological cells , vitamins , hormones , and the like . The term “ bio 

marker " indicates a biomolecule that is associated with a 

specific state of a biological environment including but not 

limited to a phase of cellular cycle , health , and disease state . The presence , absence , reduction , upregulation of the bio 

marker is associated with and is indicative of a particular state . The term “ biological environment ” refers to any biological setting , including , for example , ecosystems , orders , families , genera , species , subspecies , organisms ,tissues , cells , viruses , organelles , cellular substructures , pri ons , and samples of biological origin . Exemplary chemical 

analytes include molecular targets such as small molecules , proteins , nucleic acids , and also cells , tissues , and organ isms . 

[ 0062 ] The term “ spectroscopic probe ” indicates a sub 

stance that is suitable to be detected based on an interaction 

between a radiation and the substance through a spectro scopic instrument .Exemplary spectroscopic probes com 

prise Raman probes and fluorescence probes . The terms 

“ Raman active molecule ” or “ Raman probe ” as used herein refer to a molecule capable having a polarization - dependent vibrational mode excited by an incident light .The vibra tional energy stored in the molecule is transformed into a scattering light corresponding to a specific frequency . In 

particular , detected signals emitted by Raman probes can 

take the form of Raman spectra . Accordingly , in Raman 

spectra for a certain Raman probe , each peak represents the vibrational frequency corresponding to resonance energy of 

the functional groups in the Raman probe as detected . Therefore , Raman spectra are intrinsic properties of the 

molecules such as a “ molecular fingerprint ” to identify the 

molecule without need to use of any additional labels . 

[ 0063 ] In some embodiments , Raman probes suitable to be 

included in the chemical analyte include Raman - active 

molecules having polarization - dependent rotational modes . Exemplary Raman probes suitable to be used in the chemical 

analyte include trans - 1,2 bis- ( 4 - pyridyl ) ethylene ( BPE ) , Cy - 3 , Cy - 3.5 , Cy - 5 , Cy - 5.5 , Cy - 7 , Rhodamine 6G ( R6G ) , methylene blue ( MB ) , 5 - carboxyfluorescein or 6 - car boxyfluorescein ( FAM ) , N , N , N ' , N ' - tetramethyl - 6 - carboxy 

rhodamine ( TAMRA ) , 6 - carboxy - 4,7,2 ' , 7 ' - tetrachlorofluo rescein ( TET ) , 6 - carboxy - Xrhodamine ( ROX ) , ( 3- ( 5,6,4,7' tetrachloro - 5 ' - methyl - 3 ' , 6 ' - dipivaloylfluorescein - 2 - yl ) propanamidohexyl - 1-0- ( 2 - cyanoethyl ) - ( N , N - diisopropyl ) ) 

Yakima Yellow® , 6 - ( ( ( 4 ( 4,4 - difluoro - 5- ( 2 - thienyl ) -4 - bora 

3a , 4a - diaza - s - indacene - 3 - yl ) phenoxy ) acetyl ) amino ) 

hexanoic acid ( BODIPY TR - X ) and additional probes iden tifiable by a skilled person upon reading of the present disclosure . 

[ 0064 ] The term “ fluorescent probe ” indicates a substance 

that is detectable through emission of a visible light by the 

substance following absorption by the same substance of light of a differing , usually nonvisible , wavelength . Exem 

plary fluorescent probes suitable in the chemical analyte include Cy - 3 , Cy - 3.5 , Cy - 5 , Cy - 5.5 , Cy - 7 , Rhodamine 6G 

( R6G ) , methylene blue ( MB ) , TAMRA , and additional 

probes identifiable by a skilled person . 

[ 0065 ] The term “ attach ” or “ attached ” refers to connect ing or uniting by a bond or other link or force that keeps two 

or more components together , which encompasses either 

direct or indirect attachment where , for example , a first 

molecule is directly bound to a second molecule or material , or one or more intermediate molecules are disposed between 

the first molecule and the second molecule or material . The 

term “ bind ” , “ binding ” , and “ conjugation ” indicates an 

attractive interaction between two elements that results in a 

stable association of the elements in which the elements are 

in close proximity to each other . Attractive interactions include both non - covalent binding and covalent binding . Non - covalent binding indicates a type of chemical bond , such as protein - protein interaction , that does not involve the 

sharing of pairs of electrons , but rather involves more dispersed variations of electromagnetic interactions . Non 

covalent bonding includes ionic bonds , hydrophobic inter 

actions , electrostatic interactions , hydrogen bonds , and 

dipole - dipole bonds . Electrostatic interactions include asso 

ciation between two oppositely charged entities . 

a 
US 2022/0325332 A1 Oct. 13 , 2022 

7 

a 

a 

) 

[ 0066 ] According to an exemplary embodiment , spectro scopic probes , such as methylene blue , can be attached to the 

chemical analyte formed by an oligonucleotide through active ester coupling to an amine group ( e.g. a 3 ' C7 amine of an oligonucleotide aptamer ) . [ 0067 ] In some embodiments , a spectroscopic probe , and 

in particular a Raman probe , can be attached to the chemical 

analyte by a covalent bond , with or without one or more 

intermediate molecules , to any position where attachment 

does not interfere with binding to the aptamer or hybridiza tion to the chemical analyte . [ 0068 ] The wording “ specific ” or “ specificity ” with refer 

ence to hybridization or binding of the chemical analyte or 

generally of a first molecule to a second molecule refers to 

the recognition , contact , and formation of a stable complex between the first molecule and the second molecule , together with substantially less to no recognition , contact 

and formation of a stable complex between each of the first 

molecule and the second molecule with other molecules that 

may be present . Exemplary specific bindings include poly nucleotide hybridization . The term “ specific ” as used herein 

with reference to a sequence of a polynucleotide refers to the unique association of the sequence with a single polynucle otide which is the complementary sequence . [ 0069 ] In some embodiment , a nucleic acid core of a DNA 

switch is immobilized through electrostatic forces to a suitable substrate so that the spectroscopic probe attached to 

chemical analyte is likewise immobilized to the nucleic acid 

core proximate to the substrate . [ 0070 ] In several embodiments , DNA switch 209 or bio 

marker signal amplifier 208 herein described can detect chemical analytes with a high sensitivity showing a limit of detection s100 PM and more particularly within a dynamic 

range spanning from about 100 PM to about 1 PM depending 

on the assay performed . [ 0071 ] In several embodiments , DNA switch 209 or bio marker signal amplifier 208 can detect chemical analytes with high specificity , wherein the selective hybridization of 

the chemical analyte to DNA switch 209 or biomarker signal amplifier 208 over other analytes can be shown by specific discrimination of the chemical analyte via electrical detec 

tion or optical detection . [ 0072 ] In an embodiment , reporter particle 201 includes a nanoparticle , a quantum dot , a charged polymer , or a com 

bination thereof . The nanoparticle of reporter particle 201 

can include a gold nanoparticle . The nanoparticle of reporter particle 201 can have a surface charge . [ 0073 ] In an embodiment , reporter particle 201 includes a spectroscopic probe such as a fluorophore , Raman probe , or 

the like disposed on a nanoparticle . [ 0074 ] In an embodiment ,chemical analyte 203 includes 

a nucleic acid , DNA , RNA , or a combination thereof . According to an embodiment , with reference to FIG . 8 , chemical analyte 203 includes aptamer 215 and analyte 216 . The analyte can include a spectroscopic probe , chemical 

functional group , and the like . [ 0075 ] In an embodiment , surface strand 204 includes single stranded DNA . [ 0076 ] In an embodiment , particle strand 206 includes a base sequence that is complementary to the single stranded 

DNA of the surface strand 204 . 

[ 0077 ] In an embodiment , analysis substrate 205 includes 

a metal , a glass , a ceramic , or a combination thereof on 

which the other components of biomarker signal amplifier 208 or DNA switch 209 can be disposed . [ 0078 ] In an embodiment of DNA switch 209 , when chemical analyte 203 is hybridized to first helix strand 211 , reporter particle 201 remains attached to nucleic acid core 

210 and is sterically or thermodynamically precluded from 

interacting with first helix strand 211 . [ 0079 ] In an embodiment , DNA nanostructure framework 

202 includes a 2D nanostructure . Exemplary 2D nanostruc 

tures include wireframes of polynucleotides , DNA origami , and the like such as 2D arrays . [ 0080 ] In an embodiment , DNA nanostructure framework 

202 includes a 3D nanostructure . Exemplary 2D nanostruc 

tures include wireframes of polynucleotides , DNA origami , and the like such as polyhedral ,bundles , and the like . According to an embodiment ,DNA nanostructure frame 

work 202 is a DNA backbone helix . 

[ 0081 ] Elements of DNA nanotechnology - based bio 

marker measurement platform 200 and its components can 

be various sizes and can be varied by a choice of materials . [ 0082 ] It is contemplated that large nanoparticles ( e.g. , with large surface charge or decorated with fluorophores and 

the like ) integrated within a DNA nanostructure framework amplify electrical signal generated in presence of a chemical 

analyte . With reference to FIG . 1 , a single - stranded DNA as 

surface strand 204 is attached to a surface of analysis substrate 205 ( e.g. , gold , silica , and the like etc. ) using a chemical attachment ( e.g. , thiol chemistry , silane chemistry , and the like ) . Reporter particle 201 ( e.g. , gold nanoparticle , quantum dot , large charged polymer , and the like ) with a 

complementary DNA strand as particle strand 206 is hybrid ized with surface strand 204 to attach reporter particle 201 

in proximate to the surface of analysis substrate 205. The 

ability to use DNA nanostructures to confine reporter par ticle 201 proximate to the surface provides a sensitive 

detection modality such as electronic detection via an elec tronic signal . [ 0083 ] Regarding DNA switch 209 , for robust signal amplification , DNA switch 209 provides selective rather 

than non - specific binding of chemical analyte 203 ( e.g. , from unintended interactions between released reporter par ticle 201 and analysis substrate 205 ) that could otherwise 

result in erroneous kinetics measurement . DNA switch 209 

minimizes non - specific interactions with chemical analyte 203. While the configuration shown in FIG . 2 include DNA 

helix bundle 218 as nucleic acid core 210 , nucleic acid core 

210 is not limited to just DNA helix bundle 218. Indeed , a 

variety of 2D and 3D DNA nanostructures for nucleic acid 

core 210 supports functions of DNA switch 209described 

here . 

[ 0084 ] In the configuration shown in FIG . 2 , nucleic acid 

core 210 is assembled with reporter particle 201 including particle strand 206 that can be a DNA strand hybridized with 

both second helix strand 212 and first helix strand 211. This 

configuration can restrict reporter particle 201 to be proxi 

mate to the surface of analysis substrate 205 where reporter particle 201 can be detected using a sensing approaches discussed below . Upon addition of chemical analyte 203 , which preferentially binds first helix strand 211 with the strand displacement shown in panel B of FIG . 1 , reporter particle 201 is displaced from being proximate to analysis substrate 205 and moves distally away from analysis sub 

strate 205 as shown in panel B of FIG . 2 , indicating bound chemical analyte 203. Advantageously , when chemical ana 
US 2022/0325332 A1 Oct. 13 , 2022 

8 

lyte 203 is bound by DNA switch 209 through hybridization not first helix strand 211 , reporter particle 201 is still 

constrained to DNA helix bundle 218 of nucleic acid core 

210. Furthermore , reporter particle 201 is sterically and thermodynamically precluded from interacting with first 

helix strand 211 , minimizing any non - specific interactions with analysis substrate 205 . [ 0085 ] In an embodiment , DNA switch 209 has tunable sensitivity toward chemical analyte 203. As unlatching of 

reporter particle 201 from analysis substrate 205 is mediated 

by binding competition between second helix strand 212 and 

chemical analyte 203 binding for first helix strand 211 

sequence , the sensitivity of the displacement of reporter particle 201 with respect to analysis substrate 205 can be 

tuned by changing the predetermined second helix strand 212 / first helix strand 211 affinity via sequence length . This 

tuning can simultaneously modify the thermodynamics ( ulti 

mate binding affinity ) and kinetics ( binding rate ) with 

respect to chemical analyte 203. As thermodynamics and 

kinetics can be measured from the same signal readout , and 

they can be characterized for chemical analyte 203 to provide an internal consistency check for detection of chemical analyte 203 . [ 0086 ] With regard to the signal readout , biomarker signal amplifier 208 and DNA switch 209 are compatible with 

multiple readout methods that include but are not limited to 

the following techniques . When reporter particle 201 with a 

large surface charge is used ( e.g. , Au nanoparticles with a high surface coverage of DNA , and the like ) with analysis substrate 205 that is connected to a charge sensitive elec tronic readout system ( e.g. , field - effect transistors , amplifi 

ers , and the like ) , a large change in the surface potential at 

the input of the electronics interface results that is easily detected . Exemplary readout systems are described in U.S. 

patent application Ser . Nos . 16 / 220,866 , 16 / 867,590 , and 

17 / 029,999 , the disclosures of which are incorporated herein 

by reference in their entirety . [ 0087 ] Signals output by each readout approach can be 

processed to extract information about the type or concen 

tration of chemical analyte 203. Because biomarker signal amplifier 208 and DNA switch 209 leverages nanoscale 

features of DNA constructs , such can be formatted into a 

sensor array to simultaneously measure a spatial distribution 

of chemical analytes 203 that can be suited for processing with the pattern recognition capabilities of artificial intelli 

gence ( AI ) or machine learning ( ML ) algorithms . Such 

approaches include but are not limited to deep neural 

networks ( DNNs ) , neuromorphic elements , or other soft 

ware or hardware components . [ 0088 ] In an embodiment ,chemical analyte 203 , bio 

marker signal amplifier 208 , or DNA switch 209 can include 

a molecular adapter , e.g. , chemical analyte 203 as shown in 

FIG . 8. Molecular adapters can be , e.g. , an aptamer , anti 

body , protein , and the like that can be conjugated to probe DNA strands for recognition of various target analytes , e.g. , analyte 216 , in solution to form chemical analyte 203. FIG . 8 shows aptamer 215 that captures target analyte 216 in 

solution and forms chemical analyte 203. When analyte 216 is bound to aptamer 215 , aptamer 215 changes conformation 

to provide aptamer exchange region 220 to be activated . The 

DNA probe strands ( e.g. , aptamer exchange region 220 ) 

attached to aptamer 215 in chemical analyte 203 interact 

with sites ( e.g. , first helix strand 211 ) on DNA helix bundle 218 of nucleic acid core 210 to displace reporter particle 

201. This modular approach provides DNA nanotechnology 

based biomarker measurement platform 200 to rapidly adapt to particular applications . DNA nanotechnology - based bio 

marker measurement platform 200 with aptamer 215 - based 

chemical analyte 203 also allows commonly used 

approaches such as sandwich antibody assays to be adapted for use with advances in biotechnology . 

[ 0089 ] Regarding DNA amplifier gain , resolution , and dynamic range , the gain of biomarker signal amplifier 208 or 

DNA switch 209 can be precisely defined by engineering the properties of reporter particle 201 ( e.g. , using geometry , surface charge , fluorophore density , and the like ) . FIG . 9 

shows DNA nanotechnology - based biomarker measurement platform 200 that operates as a multi - chemical analyte 

sensor with a variable gain at each interaction site 209.k , defined by the size and charge of individual reporter par ticles 201. Each analyte binding site of DNA switch 209.k is 

designed to bind a different type of chemical analyte 203. All 

binding sites 209 are read with a single electronic detector . When chemical analytes 203 bind to the individual sites 

209.k at individual first helix strand 211 , the change in the 

signal ( shown in FIG . 9 as AA , AB , AC , and the like ) are proportional to the size and charge on the respective reporter particle 201 , e.g. , particle A , B , C , and the like . When the 

electronic detector has single particle resolution , the detec tion of binding events allows determination of the specific site 209 that was activated and therefore the type of chemical analyte 203 

[ 0090 ] DNA nanotechnology - based biomarker measure 

ment platform 200 provides a one - to - one correspondence 

between the detection of chemical analyte 203 and its amplification through displacement of reporter particle 201 

from analysis substrate 205. The resolution of the measure 

ment is determined by the sensitivity of the detection technique to reporter particle 201. As an example , electro static interfacial potential § of a hydrated nanoparticle covered with DNA strands is tens of millivolts so that single molecule discrimination of reporter particles 201 and single molecule detection of bound chemical analyte 203 can be 

performed . A similar resolution is obtained by using optical and other imaging techniques to measure reporter particles 201 such as fluorescent nanoparticles , quantum dots and the 

like . 

9 

a 

[ 0091 ] It is contemplated that agile nucleic acid sensor 

array 113 with agile nucleic acid sensors 200 can be con 

figured in various manners . In an embodiment , substrate 101 

supports the CMOS circuitry in n contact with a pixel element 102. The pixel element 102 includes sensor working electrode 104 made from a conductive material in electrical 

contact with the gate of a transistor 105 and in electrical 

contact with fluid surrounding the pixel . The transistor is 

powered by a drain voltage source 106 , and the current in the channel is interrogated using a transimpedance amplifier 107. Here , the output of the transistor is communicated electrically to signal processor 103 that includes analog to 

digital converters , multiplexers and memory registers to 

configure the measurements . The pixel element 102 also can 

include a sensor working electrode 108 that is biased by voltage follower 109 and is in electrical contact with fluid surrounding the pixel . The voltage follower 109 can be biased by a voltage set - point source 110 and a sensor 

reference electrode 108 that provide a chemical reference 

that is in electrical contact with fluid surrounding the pixel . 
US 2022/0325332 A1 Oct. 13 , 2022 

9 

a 

2 

[ 0092 ] In some embodiments , transimpedance amplifier 201 is a readout for sensor working electrode 205 and is in 

electrical communication with signal processing element 

103 in an absence of transistor 105 . 

[ 0093 ] Sensor working electrode 205 can be functional 

ized with chemical species that control its behavior and 

allow the measurements of chemical analytes . In an embodi 

ment , agile nucleic acid sensor 200 includes working elec 

trode 205 in contact with a DNA switch 209 and that 

supports first helix strand 211 made from a DNA sequence , an aptamer or other suitable molecular adapter , reporter particle 201 including gold particles covered in DNA , DNA 

nanostructures or other structures constructed from charged species to amplify movement of reporter particle 201 closer 

to or further away from analysis substrate 205. In an analyte off configuration ( FIG . 3A ) , reporter particle 201 is held in 

place by first helix strand 211. In the analyte on configura tion ( FIG . 3B ) , reporter particle 201 is released from first 

helix strand 211 by chemical analyte 203 and moves away from analysis substrate 205. The reporter particle 201 is 

attached to nucleic acid core 210 and moves reversibly between the analyte off and analyte on configurations . [ 0094 ] In an embodiment , agile nucleic acid sensor array 113 includes a plurality of agile nucleic acid sensors 200 , and agile nucleic acid sensor array 113 includes a CMOS chip with integrated circuitry and electrodes ( e.g. , sensor 

counter electrode 108 , analysis substrate 205 , sensor refer 

ence electrode 111 ) that interface to DNA switch 209 via 

chemical functionalization of the electrodes in an array of 

agile nucleic acid sensors 200 that measure various bio 

markers . FIG . 1 and FIG . 2 show embodiments of CMOS circuitry , or agile nucleic acid sensor array 113 and agile nucleic acid sensor 200 can include other configurations and 

electronics . The surface functionalization leverages DNA 

origami nanostructures of DNA switch 209. reporter particle 201 allows a weak signal upon binding of chemical analyte 203 to first helix strand 211 to be amplified . The degree of amplification determines the sensitivity of agile nucleic acid 

sensor array 113 and can be tuned by the size and charge on 

the reporter particle 201 to allow measurements of 

ensembles of chemical analyte 203 , e.g. , down to a single chemical analyte 203 with a signal to noise ratio ( SNR ) greater than 20 dB . 

[ 0095 ] The first helix strand 211 allows the strength of the interaction with the chemical analyte 203 to be tuned via 

sequence mismatches , which alter the thermodynamics of 

the pair and thereby the kinetics . These engineered weak 

interactions will allow the chemical analyte 203 to trigger reporter particle 201 to the analyte on position and revers ibly return reporter particle 201 to the analyte off position . The measurand is the rate at which the chemical analyte 203 

attaches to first helix strand 211 and the rate at which 

chemical analyte 203 dissociates therefrom . These rate con 

stants can be tuned to vary between milliseconds to minutes 

and are determined by the strength of the interaction 

between chemical analyte 203 and first helix strand 211 . [ 0096 ] The nucleic acid core 210 ( e.g. , DNA origami ) provides a supporting structure to anchor the first helix 

strand 211 and reporter particle 201. The nucleic acid core 

210 can be a six - helix bundle , e.g. , as shown in FIG . 3 , or 

another geometric form and can be constructed from DNA 

or another polymer that selectively attaches chemical ana 

lyte 203. The nucleic acid core 210 is a stable structural 

element for the other components of DNA switch 209 and is 

an addressable member for disposition of first helix strand 

211 , e.g. , via the sequence of nucleic acid core 210. This 

allows different versions of nucleic acid core 210 to be 

altered or paired with different versions of first helix strand 

211 in DNA switch 209. In this manner , various agile nucleic 

acid sensors 200 in agile nucleic acid sensor array 113 independently can have different or identical agile nucleic 

acid sensors 200. This modularity allows each analysis substrate 205 of the CMOS array to be customized via a 

different sequence of first helix strand 211. Therefore , each 

element of agile nucleic acid sensor array 113 can provide a 

different response to chemical analyte 203 having the same 

sequence and provide a fingerprint that identifies chemical 

analyte 203 . [ 0097 ] With reference to FIG . 6 and FIG . 7 , agile nucleic 

acid sensor array 113 can be made with , operated with , or 

include fluidics , e.g. , microfluidics . Here , microfluidic lines 

are in fluidic communication with substrate 101. During formation of agile nucleic acid sensor array 113 , the micro 

fluidic lines are disposed on substrate 101 such that specific types of agile nucleic acid sensors 200 are formed in selected 

areas of substrate 101. During operation of agile nucleic acid 

sensor array 113 , the microfluidics lines can be fluidically interconnected to one another such that the individual micro fluidics lines are distributed the same composition to every agile nucleic acid sensor 200 in the agile nucleic acid sensor 

array 113. Similarly , selected microfluidics lines can flow 

different compositions in an on - demand , manual , or pre programmed way . The agile nucleic acid sensor array 113 

can include fluidic ports on a surface of substrate 101 ( e.g. , a top , side , or bottom surface ) to pump a composition that 

can include chemical analyte 203 through the microfluid 

layer that include the microfluidic lines . Electrical connec 

tion to elements of the microfluidic lines can be made via 

pogo pins that are aligned and pressed against electrical pads 

on substrate 101. During operation , these can be used for 

sensing . During fabrication , they can be used for electro 

phoresis to assist in depositing the agile nucleic acid sensors 

200. The pogo pins can connect to a circuit board for communicating electrical control signals or acquiring data . The circuit board can be used for deposition during making 

the agile nucleic acid sensor array 113 or for sensing . Electrical components to operate agile nucleic acid sensor 

array 113 can be included on the circuit board . [ 0098 ] The substrate 101 can be mounted in a package that 

can form a fluid - tight seal , e.g. , by compressing an elastomer 

such as an O - ring . Compression of the elastomer can occur , e.g. , by screws , springs , or the like . Compression of the parts 

can provide adequate force for electrical contact to the pogo pins . 

[ 0099 ] With reference to FIG . 6 , microfluidic package 501 

can have fabrication configuration fluid delivery 502 , wherein various supply reservoirs ( left - hard part in FIG . 6 ) provide selected compositions to sensors in agile nucleic 

acid sensor array 113 to form specific agile nucleic acid 

sensor 200. With reference to FIG . 7 , microfluidic package 501 can have sensing configuration fluid delivery 503 , wherein a supply reservoir ( left - hard part in FIG . 7 ) provides 

a composition with chemical analyte 203 to sensors in agile nucleic acid sensor array 113 to form specific agile nucleic 

acid sensor 200 . 

[ 0100 ] The top of microfluidic package 501 can include a 

top that with fluidic ports used to connect the microfluidic 

lines , screws holes to compress the microfluidic package 

a 

2 
US 2022/0325332 A1 Oct. 13 , 2022 

10 

a 

a 

501 and substrate 101 , and the like . This provides a fluidic 

seal and electrical connections . The microfluidic layer of 

microfluidic lines can be disposed under the top surface of 

microfluidic package 501 and can be in direct fluidic contact with substrate 101. An O - ring can be interposed between 

substrate 101 and the microfluidic layer to provide a her 

metic seal . The substrate 101 can include through silicon vias to allow components to be disposed on opposing surfaces thereof , e.g. , for electrical contact pads to be 

disposed on the underside of substrate 101 and agile nucleic acid sensor 200 to be disposed on top of substrate 101 to 

interface with the microfluidics . The bottom of microfluidic 

package 501 can include pogo pins as electrical conductors 

and for electrical connections to substrate 101. Pogo pins 

can be disposed on the underside to interface with a circuit 

board . The circuit board can include the electrical compo 

nents needed to operate agile nucleic acid sensor array 113 . The microfluidic package 501 can also include members for fasteners , e.g. , mounting holes to receive a fastener ( e.g. , a 

screw ) so that microfluidic package 501 can be mounted 

( e.g. , screwed ) to the circuit board . [ 0101 ] The microfluidic package 501 can be made in 

various ways , e.g. , 3D printed or machined . The microflu idics layer can be incorporated into the top of microfluidic package 501 as part of the 3D printing or machining process 

or can be a separate layer that is interposed between sub 

strate 101 and the top . The microfluidics layer can be 3D 

printed or machined . Alignment pins can be used to align an 

assemble the components of microfluidic package 501 to 

substrate 101 . 

[ 0102 ] Elements of agile nucleic acid sensor array 113 can 

be various sizes and can be made of a material that is 

physically or chemically resilient in an environment in 

which agile nucleic acid sensor array 113 is disposed . Exemplary materials include a metal , ceramic , thermoplas tic , glass , semiconductor , and the like . The elements of agile nucleic acid sensor array 113 can be made of the same or different material and can be monolithic in a single physical body or can be separate members that are physically joined . [ 0103 ] Agile nucleic acid sensor 200 and agile nucleic 

acid sensor array 113 can be made in various ways . It should 

be appreciated that agile nucleic acid sensor 200 and agile nucleic acid sensor array 113 can include a number of optical , electrical , or mechanical components , wherein such 

components can be interconnected and placed in communi 

cation ( e.g. , optical communication , electrical communica 

tion , mechanical communication , and the like ) by physical , chemical , optical , or free - space interconnects . The compo nents can be disposed on mounts that can be disposed on a 

bulkhead for alignment or physical compartmentalization . As a result , agile nucleic acid sensor 200 and agile nucleic 

acid sensor array 113 can be disposed in a terrestrial envi 

ronment or space environment .Elements of agile nucleic 

acid sensor 200 and agile nucleic acid sensor array 113 can 

be formed from silicon , silicon nitride , and the like although other suitable materials , such ceramic , glass , or metal can be 

used , the electrical conductivity of which can be selected according to operation of agile nucleic acid sensor array 113 . According to an embodiment , the elements of agile nucleic 

acid sensor 200 and agile nucleic acid sensor array 113 are 

formed using 3D printing although the elements of agile nucleic acid sensor 200 and agile nucleic acid sensor array 113 can be formed using other methods , such as injection molding or machining a stock material such as block of 

material that is subjected to removal of material such as by cutting , laser oblation , and the like . Accordingly , agile nucleic acid sensor 200 and agile nucleic acid sensor array 113 can be made by additive or subtractive manufacturing . In an embodiment , elements of agile nucleic acid sensor 200 

are selectively etched to remove various different materials 

using different etchants and photolithographic masks and 

procedures . The various layers thus formed can be subjected to joining by bonding to form agile nucleic acid sensor 200 

and agile nucleic acid sensor array 113 . 

[ 0104 ] The process for making agile nucleic acid sensor 

200 also can include forming a DNA nanostructure , e.g. , for 

DNA switch 209 , which can include selecting a mechanism for signal amplification ( surface charge , capacitance , etc. ) and surface binding ( gold / thiol , biotin / streptavidin , etc. ) of 

chemical analyte 203 by first helix strand 211 and reporter particle 201 ; determining a 3D shape of DNA origami of 

nucleic acid core 210 for sampling on / off binding states associated with signal amplification of contact of DNA switch 209 with chemical analyte 203 as well as positions for surface binding and analyte binding that will lock the 

shape in the on state ; routing viral DNA scaffold through 3D 

shape ; filling in staple strands ; truncating to lengths com 

mensurate with desired DNA synthesis technique ; and label 

ing relevant staple positions associated with surface binding and analyte binding for modification with the appropriate chemical moieties . 

[ 0105 ] The process for making agile nucleic acid sensor 

array 113 also can include forming DNA switch 209 by : combining a viral DNA scaffold , pH buffering components , divalent cations , DNA oligomer staples that fold the body of 

the nanostructure , and staples that bind the nanostructure to 

analysis substrate 205 , and staples for first helix strand 211 , wherein scaffold and staples can be present in a ratio , e.g. , from 1 : 5 to 1:10 or higher ; annealing the composition to 

remove secondary structure , e.g. , thermal annealing via heating the composition to 80 ° C. for denaturing followed by cooling to 25 ° C. at a cooling rate , e.g. , of 1 ° C./min , or alternatively chemical annealing via dialysis against a for 

mamide gradient ; labeling relevant staple positions associ 

ated with surface binding and analyte binding for modifi 

cation with the appropriate chemical moieties ; removing 

excess staples , e.g. , via PEG precipitation or molecular weight cutoff spin filter protocols ; performing quality con 

trol ( e.g. , gel electrophoresis , microscopy , and the like ) ; and 

disposing DNA switch 209 on analysis substrate 205 via a 

protocol that is suitable for the surface binding chemistry being used . 

[ 0106 ] The process for making agile nucleic acid sensor 

array 113 also can include microfluidically delivering indi 

vidual DNA switches 209 to individual agile nucleic acid 

sensors 200 by : forming microfluidic package 501 that holds 

substrate 101 and microfluidic layers ( 502 and 503 ) such 

that substrate 101 and microfluidic layers can be disas 

sembled and interchanged ; configuring the microfluidic 

package 501 in the first instance for sensor fabrication by disposing the fabrication microfluidic configuration 502 on 

substrate 101 within microfluidic package 501 ; delivering different DNA switches 209 , aligning and binding DNA 

switches 209 to predetermined sensor working electrodes 

205 ; functionalizing selected regions of substrate 101 with 

different sensor types delivered via separate microfluidic 

lines ; and removing substrate 101 from microfluidic package 
US 2022/0325332 A1 Oct. 13 , 2022 

11 

2 

2 2 

501 and disposing in a new microfluidic package 501 that contains microfluidics in the sensing configuration 503 for 

sensor operation . [ 0107 ] Agile nucleic acid sensor 200 has numerous advan 

tageous and unexpected benefits and uses . In an embodi 

ment , a process for measuring a biomarker includes : dis 

posing substrate 101 ( that includes agile nucleic acid sensor 

array 113 ) in microfluidic package 501 ; providing a micro 

fluidic fabrication fluid delivery layer 502 on substrate 101 ; delivering differently tuned DNA nanotechnology sensors to individual sensor working electrodes 205 to configure the sensing array for a selected application ( e.g. , sensing a particular chemical analyte 203 ) ; replacing layer 502 with 

operation fluid delivery microfluidic layer 503 ; delivering a composition including chemical analyte 203 sample to the 

sensor array 113 ; reading out sensing signals using interface 

pads 112 ; and analyzing the sensing signals . [ 0108 ] In an embodiment , with reference to FIG . 9 , FIG . 10 , and FIG . 11 , a process for measuring a biomarker with 

an agile nucleic acid sensor array 113 includes : operating the 

agile nucleic acid sensor array 113 that includes : a plurality of agile nucleic acid sensors 200 arranged in array and that 

individually include : a DNA switch 209 ; an analysis sub 

strate 205 in electrostatic communication with the DNA 

switch 209 that is disposed on the analysis substrate 205 ; a 

transduction member 114 in electrical communication with 

the analysis substrate 205 ; a sensor counter electrode 108 in electrical communication with and capacitively coupled to 

the analysis substrate 205 ; a sensor reference electrode 111 

in electrical communication and capacitively coupled to the analysis substrate 205 ; and a voltage follower 109 in elec 

trical communication with the sensor counter electrode 108 

and the sensor reference electrode 111 ; for individual agile nucleic acid sensor 200 in the agile nucleic acid sensor array 113 : producing a counter electrode voltage 117 by the 

voltage follower 109 ; subjecting the sensor counter elec 

trode 108 to the counter electrode voltage 117 from the 

voltage follower 109 ; contacting the DNA switch 209 with 

a chemical analyte 203 comprising the biomarker ; produc ing , by the analysis substrate 205 , a biomarker electrical 

signal 115 in response to the chemical analyte 203 contact 

ing the DNA switch 209 ; receiving , by the transduction 

member 114 , the biomarker electrical signal 115 from the 

analysis substrate 205 and producing a transduction signal 116 from the biomarker electrical signal 115 ; determining the impedance of the analysis substrate 205 from the trans 

duction signal 116 ; estimating kinetic rate constants for each 

contact between the chemical analyte 203 and the DNA 

switch 209 ; and combining the kinetic rate constants and producing a mean kinetic rate constant with uncertainty quantification for the kinetic rate constants ; aggregating the 

mean kinetic rate constants for the plurality of agile nucleic acid sensor 200 in the agile nucleic acid sensor array 113 ; and producing a kinetic fingerprint for the chemical analyte 203 from the mean kinetic rate constants . 

[ 0109 ] With further reference to FIG . 9 , FIG . 10 , and FIG . 11 , the process for measuring a biomarker with agile nucleic 

acid sensor array 113 can include reading the kinetic fin 

gerprint from the agile nucleic acid sensor array 113 ( step 120 ) ; providing the kinetic fingerprint as input to a machine learning model for classification ( step 121 ) ; providing the 

kinetic fingerprint as input to an Al - enhanced simulator 127 

( step 122 ) ; updating training data for the Al - enhanced 

simulator 127 machine learning models ( step 123 ) ; provid 

ing a physical model of the agile nucleic acid sensor 200 and 

kinetic fingerprint measurement to the Al - enhanced simu 

lator 127 machine learning models ( step 124 ) ; combining the kinetic fingerprint ,physical models , and parameter uncertainties using machine learning model to generate physics enhanced training data with uncertainty quantifica tion ( step 125 ) ; and combining Al - enhanced simulator train ing data with the sensor array kinetic fingerprint by analyte classification machine learning to produce analyte classifi 

cation with uncertatinty quantification ( step 126 ) . 

[ 0110 ] The agile nucleic acid sensor array 113 includes dynamic and tunable DNA nanotechnology sensor arrays with 10 - fold better LOD ( ~ 10 molecules ) than PCR , data 

driven modeling , Al classification , UQ , and training data and assay development for detection and classification of 

chemical analyte 203 of unknown identity . 

[ 0111 ] It should be appreciated that agile nucleic acid 

sensor array 113 is a dynamic and tunable sensor array . Randomly distributed moieties that bind an ensemble of 

known analytes and produce a signal that monotonically changes with analyte concentration ( FIG . 19 ) underlie con 

ventional bioassays . In contrast , the agile nucleic acid sensor 

array 113 includes dynamic DNA nanostructures , e.g. , agile nucleic acid sensor 200 , that allow the strength and duration 

of the interactions between agile nucleic acid sensors 200 and a target nucleic acid to be precisely defined ( FIG . 18 ) . The agile nucleic acid sensor array 113 produces a series of 

weak interactions between chemical analyte 203 and mul tiple agile nucleic acid sensors 200 in agile nucleic acid 

sensor array 113. The sum of which results in a unique molecular signature . Changes in the nucleic acid sequence ( e.g. , from mutant strains ) modify the interactions between 

the chemical analyte 203 and agile nucleic acid sensors 200 , altering the binding with individual agile nucleic acid sen 

sors 200 and changing the molecule signature . 

[ 0112 ] The agile nucleic acid sensor array 113 includes 

agile nucleic acid sensor 200 with a level of detection ( LOD ) of ~ 10 molecules , 2104 molecules , or better than conven 

tional measurements , e.g. , electrochemical detection , fluo 

rescence , and the like . This LOD is achieved by designing DNA nanostructures 209 with in - situ reporter particles 201 

that enhance the electrostatic potential output by the agile nucleic acid sensor 200 ( FIG . 18 ) and leverages the strand displacement technique to reversibly distinguish the bound 

and unbound states of chemical analyte 203 chemical ana 

lyte 203. The result is engineered detectors ( FIG . 18 ) with precise interaction kinetics defined by the DNA sequence and with tunable gain ( e.g. , greater than 104 ) via particle size 

to allow label - free and PCR - free measurements . 

[ 0113 ] The agile nucleic acid sensors 200 ordered in agile nucleic acid sensor array 113 are configured for highly multiplexed measurements and to minimize variation in 

detection of chemical analyte 203 among agile nucleic acid 

sensors 200. For precise fabrication , alignment of agile nucleic acid sensor 200 during fabrication can be achieved 

by different methods . Electrophoresis can be used , wherein 

the DNA based sensors 200 are aligned to an applied electric 

field . The electrodes for alignment can be incorporated in the 

substrate 101 or can be external . Alternatively , flow in 

microfluidic channels can be used , wherein the flow can align the sensors 200. Flexibility can be built into by including swappable microfluidic layers , wherein one design 

? 
US 2022/0325332 A1 Oct. 13 , 2022 

12 

model forms and training data improves transformation and 

representation of the measurands and UQ . [ 0127 ] With regard to training Data and multiplexed mea 

surements by , training data is used for AI classification of 

signals from the sensor array 113. Here , Al improves the 

physical description of sensors 200 and uses this improved reduced order description to train a classifier such as a DNN . A series of experiments under varying sensor parameters is 

used to generate data to train the reduced order AI models . DNA nanostructures 209 , informed by the AI , are then be 

designed to detect standard reference materials ( SRMs ) with 

> 95 % confidence in the presence of confounding molecules 

from a viral multiplex reference panel , e.g. , as described in 

Viral Multiplex Reference 11/242 , available from the 

National Institute for Biological Standards and Control at https://www.nibsc.org/products/brm_product_catalogue/de 

tail_page.aspx ? catid = 11 / 242 ( as of 2022 ) . Final AI training data is generated by systematically sampling system param 

eters ( FIG . 18 ) within limits determined by the model . This , in turn , allows the PCR - free detection of challenge samples , e.g. , including novel SARS - CoV - 2 variants in presence of 

background molecules without prior training of the AI . [ 0128 ] The articles and processes herein are illustrated further by the following Examples , which are non - limiting . 

EXAMPLE 1 

for the microfluidic layers is used during fabrication and then replaced with a separate design that is optimized for 

operation of the sensor .[ 0114 ) Advantageously , agile nucleic acid sensor array 

113 includes a high resolution electronic readout of sensor 

200 with field - effect transistors ( FETs ) for measurement of 

dynamic quantities shown in FIG . 18. These can include ( a ) the sensor type and sequence from the signal amplitude , ( b ) the association rate to allow estimation of analyte concen 

tration , and ( c ) the interaction strength from the dissociation 

rate . These multi - variate measurands , combined across all 

sensors 200 , provide the Al - enhanced simulator 127 data 

that is labelled for classification . [ 0115 ] Optimization of the sensors 200 provide mitigation of entropic penalty of reporter particle 201 that can hinder 

multiplexing , by : 

[ 0116 ] optimizing binding kinetics through control of 

the DNA nanostructure 202 sequence ; 

[ 0117 ] including of cycles of PCR to improve the sig 

nal - to - noise - ratio ( SNR ) ; 

[ 0118 ] varying the counterion concentration to control 

electrostatic interactions between the DNA strands ; 

[ 0119 ] altering sensor temperature to provide energetic 

control of the kinetic interaction ; 

[ 0120 ] metering probe - target concentrations to vary sensitivity ; 

[ 0121 ] delivering , by microfabrication and microfluid 

ics , different DNA structures , each with predetermined tunability , to separate regions of substrate 101 ; 

[ 0122 ] using microfabrication to dispose individual sen 

sor elements to minimize crosstalk between measure 

ment channels ; or 

[ 0123 ] using modified DNA / RNA bases to modify inter - strand interactions . 

[ 0124 ] Data acquired , e.g. , by , can be subjected to AI 

modeling to determine uncertainty . The DNA sensor array 113 can interact with an AI framework for robust data 

reduction . Conventional analyses iteratively solve a physical model while varying the input parameters to approximate a 

measured signal , but this can be computationally expensive and can involve the solution of partial - differential equations ( PDEs ) that describe an exponential number of reaction diffusion processes due to competing binding interactions . Moreover , relying on purely mathematical models that do 

not capture the underlying physics can produce overfitting 

and high uncertainties . [ 0125 ] With agile nucleic acid sensor array 113 , the data 

driven and hierarchal process for measuring a biomarker 

includes AI elements that reduce order modeling and involve 

forward solutions of a physical model , based on design of a 

measurement to train a deep neural network ( DNN ) that characterizes the relationship between model inputs , e.g. , binding affinities and competitive effects , and outputs , e.g. , FET voltages . [ 0126 ] Subsequently , experimental training data is used to 

optimize the AI models and identify the dominant competi tive effects to determine the classifier for a given analyte . To verify model assumptions , algorithms are tested on a suite of 

synthetic data and later with measured data . Because the 

DNN approximates model solutions for a continuum of 

conditions that occupy a high - dimensional space , the pro 

cess selects the most promising approach after testing dis cretization schemes with experimental data . Developing the 

[ 0129 ] The agile nucleic acid sensor array 113 upends conventional wisdom of requiring perfect sequence comple mentarity for robust detection . With reference FIG . 16 , agile nucleic acid sensor array 113 includes agile nucleic acid 

sensors 200 that include DNA switch 209 that are interro 

gated electronically and measure with a sufficient LOD to eliminate need for PCR enrichment .Instead of designing 

sensors optimized for a known target , agile nucleic acid 

sensors 200 in agile nucleic acid sensor array 113 can be 

heterogenous detectors whose interactions with chemical 

analyte 203 produce a series of weak interactions . The composite of these interactions produce complex , high dimensional signals that are ideal for processing with a 

properly trained Al framework , for highly multiplexed mea 

surements . The sensor array can be tuned for different 

purposes and circumstances in the field by importing dif 

ferent training of the AI . [ 0130 ] The AI here excels at extracting information from signals arising from a complex combination of competing physical processes , some of which obscure the measurand . When the dominant physical processes and their interplay 

are difficult to describe a priori , AI techniques succumb to 

either model errors or overfitting , which increase uncer tainty . This data - driven approach addresses this problem by first inverting the typical role of AI to identify physical 

processes that dominate a measurement . This facilitates AI 

tools to accurately classify the data output by the sensors 

with a quantitative measure of the associated uncertainties . 

COMPARATIVE EXAMPLE 1 

2 

[ 0131 ] With reference to FIG . 17 , conventional nucleic acid diagnostic measurements rely on sequence complemen tarity ( the canonical pairing of G to C or A to T over a region of DNA ) . This property has been the foundation of medical 

testing for decades . It underlies quantitative PCR ( PCR ) , the conventional method in viral detection that can distin 

guish analytes with subtle differences , e.g. , to separate 
US 2022/0325332 A1 Oct. 13 , 2022 

13 

distinct strains of seasonal influenza from the SARS coro 

navirus , with a limit - of - detection ( LOD ) of 100 molecules . However , PCR techniques are hindered by low multiplexing ( e.g. , qPCR is limited to the simultaneous detection of up to five regions of the genome in a single test ) . Sequence complementarity is also central to DNA microarrays , used to 

screen for mutations that underlie cancer sub - types , which 

allow multiplexed detection of hundreds of thousands of 

known sequences , albeit with LOD > 10 % molecules . Simi 

larly , other sequencing schemes allow highly multiplexed measurements but require large amounts of sample and a 

complex analysis pipeline . In these conventional cases , a specific target region of interest must be known a priori , and 

the assays optimized to achieve reliable detection . More 

over , sequence mismatches due to assay design errors cause inadequate PCR amplification or incomplete binding in 

DNA microarrays , resulting in test failures or inconclusive results . The strong dependence of assay performance on 

sequence complementarity hinders the application of con 

ventional tests to evolution scenarios ( e.g. , genetic muta tions arising from cancer sub - types or rapidly mutating viruses ) such that new assays have to be developed for each 

variant . 

[ 0136 ] The actuation of the notched rectangle origami was confirmed by adding an analyte that had a complementary 

sequence to the lock strand of first helix strand 211 ( FIG . 8 ) . The actuation was verified by measuring the change in the 

capacitance as a function of time at the surface of the 

working electrode at 100 Hz as shown in FIG . 13. The plot shows the aggregate response of all structures on the surface . Assuming a first order response , the kinetic association time 

was determined to be ~ 8 minutes . [ 0137 ] The reprogrammability of the method was verified by competitively dissociating the analyte from the lock 

strand using the strand displacement technique . An antidote 

strand that had a high affinity for the analyte was used to 

dissociate the strand and return the structure to its analyte off 

position on the surface of the electrode as shown in FIG . 14 . The ability to measure the kinetic association and dissocia tion of an origami structure provides the basis for repro grammability of the sensor array and will enable scaling for 

highly multiplexed measurements . 

EXAMPLE 3 

EXAMPLE 2 

[ 0132 ] FIG . 8 shows an exemplary design and fabrication 

of a nucleic acid origami structure for DNA switch 209. The 

notched rectangle origami design process ( FIG . 8 , middle 

panel ) shows the M13 scaffold DNA and the location of the 

staple strands that bind the scaffold to allow the final shape . The structure was verified using AFM imaging ( FIG . 8 , top panel ) . The structures are held by thiol bonds to a gold surface ( blue dots ) and designed to actuate by 6.6 nm away from the surface upon binding of an analyte to the lock 

strand ( red dot ) . The movement of the highly charged origami away from the surface provides charge amplifica tion similar to a gold nanoparticle covered with DNA or 

other charged entities . [ 0133 ] The kinetics of the switching behavior are con trolled by the sequence of the lock motif and can be tuned by introducing different lock strand sequences . A key idea is 

that the lock strand is highly customizable and modular 

since the DNA origami scaffold provides an addressable way of customizing this first helix strand 211 sequence without altering the functionality of the rest of the structure . There 

fore this approach can be used to generate a multitude of 

DNA - origami structures that provide a different response to 

one or more chemical analytes 203 . [ 0134 ] The functionalization of the working electrode 205 

was quantified using electrical measurements of the surface capacitance shown in FIG . 12. The capacitance measure 

ments were performed in electrolyte solution as a function of 

the excitation frequency of an applied sinusoidal AC volt 

age . 

[ 0135 ] For the bare electrode surface , a high double layer capacitance was measured . Upon adding the notched rect 

angle origami this capacitance dropped drastically ( > 80 % at 

100 Hz ) indicating a significant increase in the thickness of 

species adsorbed to the surface . The addition of the origami to the surface resulted in an effective circuit model that had 

two capacitors in series as shown in FIG . 2 ( bottom right ) , and the density of notched rectangle origami on the surface 

was 1.2x100 cm - 2 or covering approximately 7 % of the 

sensor surface . 

[ 0138 ] Multiplexed biomarker measurements distinguish analytes with subtle but important differences , e.g. , separat ing distinct strains of seasonal influenza from the SARS 

coronavirus . The measurements can detect several types of genetic mutations that underly cancers . In all cases , the development of a new assay is slow , expensive and error 

prone . 

[ 0139 ] The are PCR - free measurements of RNA or DNA 

at extremely low copy numbers , with data analysis per formed with AI . The sensors 200 include electronic detec 

tors , and each includes DNA switch 209 whose sequence complementarity for an analyte determines the energetics of 

the interaction . This energy barrier controls both the analyte binding affinity via the thermodynamics and the interaction kinetics . Because many detectors populate a given sensor , competitive interactions between them lead to complex , high - dimensional signals that are ideal for processing with 

an Al framework . The AI implements a rich mathematical 

structure that allows the flexible sensors to be recalibrated 

on - the - fly ( via reference data ) to detect new analytes , e.g. , those associated with emerging diseases . Moreover , this approach can be adapted to existing measurements , such as 

DNA microarrays resulting in even broader impact . [ 0140 ] The AI here provides reduced - order modeling . AI 

excels at extracting information from signals arising from a complex combination of competing physical processes , some of which obscure the measurand . This ability is predicated on having a model that is sufficiently rich to 

accurately describe these underlying processes . However , when the dominant physical processes and their interplay are difficult to describe efficiently a priori , AI techniques suc 

cumb to either model errors or overfitting , both of which 

increase uncertainty . The uses a data - driven process that 

overcomes this problem by inverting the role of AI , by using it to first identify physical processes that dominate a mea 

surement . This allows for efficient characterization of the 

measurement processes and for downstream AI tools to 

accurately characterize the measurements . [ 0141 ] With regard to uncertainty , incorporation of uncer 

tainty quantification ( UQ ) within an Al framework is chal 

lenging . Here , the Al includes forward and inverse modeling of the high - dimensional , multi - scale , and multi - physics parameter space associated with our measurements . The 
US 2022/0325332 A1 Oct. 13 , 2022 

14 

analysis yield a quantitative understanding of uncertainties 

and inadequacies in mathematical models , resulting in a new 

stochastic design paradigm for biomarker measurements . [ 0142 ] Training data is used . Adding an additional com posite layer of optimization to allow UQ increases reliance 

of Al models on the quality and quantity of training data . The agile nucleic acid sensors 200 can generate large and comprehensive reference measurements tailored for AI . A 

defined sequence mismatch between the DNA nanostruc 

tures 209 and measured nucleic acid analytes 203 allows 

affinity and kinetics of the interactions to be varied system atically to generate unique high - dimensional training data . [ 0143 ] Sensors 200 that combine DNA nanotechnology with sensitive electronics are validated with genetic SRMs 

measured using quantitative PCR ( qPCR ) and digital PCR 

( dPCR ) measurements . The feedback loop resulting from using the DNA nanodevices 200 to generate the large amount of high - quality training data needed to develop the 

Al models , which in turn results in robust biometrology ( FIG . 11 ) . [ 0144 ] Data analyses can iteratively solve a model of a 

physical system while varying input parameters to match the 

output with an acquired signal and deduce optimal informa 

tion about a measurand . This approach can be computation ally expensive for use with physical models of our sensor 

array , which includes partial - differential equations ( PDEs ) 

that describe many reaction - diffusion processes combinato rially due to competitive binding interactions . Relying solely 

on mathematical models that do not capture the underlying physics can result in overfitting and high uncertainties . Therefore , and measuring a biomarker involves a hierarchal 

framework that leverages AI for reduced order modeling and 

includes forward solutions of a physically - informed model 

once upon design of a measurement . A deep neural network 

( DNN ) is trained to characterize the relationship between model inputs , e.g. , binding affinities and competitive effects , and outputs , e.g. , voltages on sensor working electrode 205 . [ 0145 ] Empirical data was used to optimize the AI model 

and identify dominant competitive effects to determine the classifier for a given chemical analyte 203. Making this 

classification scheme includes selection of basis functions that : approximate the underlying physical processes through 

the construction of a loss function , reduce dimensional complexity of the model , allow efficient training and infor 

mation extraction , and estimate errors associated with ignor ing non - dominant effects . [ 014 ] To verify model assumptions , the algorithms are 

tested on a suite of synthetic data and later with measured 

data . Because the DNN approximates solutions to our model 

for a continuum of conditions that occupy a high - dimen 

sional space , our approach allows us to select the most promising approach after testing a myriad discretization 

schemes with the measured data . This step can be performed iteratively with the sensor development for optimal mea 

surements . 

[ 0147 ] Developing the appropriate model forms and train 

ing data allow improved transformation and representation of the measurands and robust UQ . Data for this are shown 

in FIG . 15 that uses affine transforms to accurately estimate qPCR uncertainty . [ 0148 ] The following are incorporated by reference herein in their entirety : 

[ 0149 ] GIS Aid . SARS - CoV2 Variants available at https://www.gisaid.org/hcov19-variants/ . 

[ 0150 ] Revealing thermodynamics of DNA origami folding via affine transformations . Nucleic Acids Res . gkaa283 ( 2020 ) doi : 10.1093 / nar / gkaa283 . [ 0151 ] Dynamic DNA nanotechnology using strand 

displacement reactions . Nat . Chem . 3 , 103-113 ( 2011 ) . [ 0152 ] An easy - to - prepare mini - scaffold for DNA ori 

gami . Nanoscale 7 , 16621-16624 ( 2015 ) . [ 0153 ] Reproducible Performance Improvements to 

Monolayer MoS2 Transistors through Exposed Mate 

rial Forming Gas Annealing . ACS Appl . Mater . Inter 

faces 11 , 16683-16692 ( 2019 ) . [ 0154 ] Rapid , quantitative therapeutic screening for 

Alzheimer's enzymes enabled by optimal signal trans 

duction with transistors . The Analyst 145 , 2925-2936 

( 2020 ) . [ 0155 ] Quantum capacitance - limited MoS2 biosensors 

enable remote label - free enzyme measurements . Nanoscale 11 , 15622-15632 ( 2019 ) . [ 0156 ] Charge detector and process for sensing a 

charged analyte . US Patent Application Publication No. 

2020/0264129 . 

[ 0157 ] Structure and Process For High - Resolution Bio 

sensor Charge Readout With Silicon Transistors . U.S. 

patent application Ser . No. 17 / 029,999 . [ 0158 ] Scalable nano - bioprobes with sub - cellular reso 

lution for cell detection . Biosens . Bioelectron . 45 , 267-273 ( 2013 ) . [ 0159 ] Diffusion - limited reactions in nanoscale elec 

tronics . Methods Appl . Anal . 26 , 149-166 ( 2019 ) . [ 0160 ] Mass spectral similarity mapping applied to 

fentanyl analogs . Forensic Chem . 100237 ( 2020 ) doi : 

10.1016 / j.forc.2020.100237 . [ 0161 ] Stochastic regression modeling of chemical 

spectra . Chemom . Intell . Lab . Syst . 139 , 26-32 ( 2014 ) . [ 0162 ] Affine analysis for quantitative PCR measure 

ments . Anal . Bioanal . Chem . 412 , 7977-7988 ( 2020 ) . [ 0163 ] Viral Multiplex Reference , available at https : // www.nibsc.org/products/brm_product_catalogue/de 

tail_page.aspx ? catid = 11 / 242 . [ 0164 ] Grand View Research . Infectious Disease In vitro Diagnostics Market Size , Share & Trends Analy sis Report By Product , By Application , By Technology ( Immunoassay , Molecular Diagnostics ) , By End - use , By Region , And Segment Forecasts , 2021-2028 , avail 

able at https://www.grandviewresearch.com/industry analysis / ivd - infectious disease - market . [ 0165 ] Letter to Dr. Eric S. Lander , the President's 

Science Advisor and nominee as Director of the Office 

of Science and Technology Policy , available at https : // www.whitehouse.gov/briefing-room/statements-re 

leases / 2021 / 01 / 20 / a - letter - to - dr - eric - s - lander - the presidents - science - advisor - and - nominee - as - director 

of - the - office - of - science - and - technology - policy / . [ 0166 ] The market trend analysis and prospects of can 

cer molecular diagnostics kits . Biomater . Res . 22 , 2 

( 2018 ) . [ 0167 ] The processes described herein may be embodied 

in , and fully automated via , software code modules executed 

by a computing system that includes one or more general 

purpose computers or processors . The code modules may be 

stored in any type of non - transitory computer - readable 

medium or other computer storage device . Some or all the 

methods may alternatively be embodied in specialized com 

puter hardware . In addition , the components referred to 
US 2022/0325332 A1 Oct. 13 , 2022 

15 

herein may be implemented in hardware , software , firm 

ware , or a combination thereof . [ 0168 ] Many other variations than those described herein 

will be apparent from this disclosure . For example , depend ing on the embodiment ,certain acts , events , or functions of 

any of the algorithms described herein can be performed in 

a different sequence , can be added , merged , or left out 

altogether ( e.g. , not all described acts or events are necessary for the practice of the algorithms ) . Moreover , in certain 

embodiments , acts or events can be performed concurrently , e.g. , through multi - threaded processing , interrupt process ing , or multiple processors or processor cores or on other 

parallel architectures , rather than sequentially . In addition , different tasks or processes can be performed by different 

machines and / or computing systems that can function together . [ 0169 ] Any logical blocks , modules , and algorithm ele 

ments described or used in connection with the embodi 

ments disclosed herein can be implemented as electronic 

hardware , computer software , or combinations of both . To clearly illustrate this interchangeability of hardware and 

software , various illustrative components , blocks , modules , and elements have been described above generally in terms of their functionality . Whether such functionality is imple mented as hardware or software depends upon the particular application and design constraints imposed on the overall 

system . The described functionality can be implemented in varying ways for each particular application , but such imple mentation decisions should not be interpreted as causing a 

departure from the scope of the disclosure . [ 0170 ] The various illustrative logical blocks and modules 

described or used in connection with the embodiments disclosed herein can be implemented or performed by a machine , such as a processing unit or processor , a digital signal processor ( DSP ) , an application specific integrated circuit ( ASIC ) , a field programmable gate array ( FPGA ) or other programmable logic device , discrete gate or transistor logic , discrete hardware components , or any combination 

thereof designed to perform the functions described herein . A processor can be a microprocessor , but in the alternative , the processor can be a controller , microcontroller , or state 

machine , combinations of the same , or the like . A processor 

can include electrical circuitry configured to process com 

puter - executable instructions . In another embodiment , a 

processor includes an FPGA or other programmable device that performs logic operations without processing computer executable instructions . A processor can also be imple mented as a combination of computing devices , e.g. , a 

combination of a DSP and a microprocessor , a plurality of microprocessors , one or more microprocessors in conjunc tion with a DSP core , or any other such configuration . Although described herein primarily with respect to digital technology , a processor may also include primarily analog 

components . For example , some or all of the signal pro cessing algorithms described herein may be implemented in 

analog circuitry or mixed analog and digital circuitry . A 

computing environment can include any type of computer system , including , but not limited to , a computer system based on a microprocessor , a mainframe computer , a digital signal processor , a portable computing device , a device 

controller , or a computational engine within an appliance , to 

name a few . [ 0171 ] The elements of a method , process , or algorithm described in connection with the embodiments disclosed 

herein can be embodied directly in hardware , in a software 

module stored in one or more memory devices and executed 

by one or more processors , or in a combination of the two . A software module can reside in RAM memory , flash 

memory , ROM memory , EPROM memory , EEPROM 

memory , registers , hard disk , a removable disk , a CD - ROM , or any other form of non - transitory computer - readable stor 

age medium , media , or physical computer storage known in 

the art . An example storage medium can be coupled to the 

processor such that the processor can read information from , and write information to the storage medium . In the alter native , the storage medium can be integral to the processor . The storage medium can be volatile or nonvolatile . [ 0172 ] While one or more embodiments have been shown 

and described , modifications and substitutions may be made 

thereto without departing from the spirit and scope of the 

invention . Accordingly , it is to be understood that the present invention has been described by way of illustrations and not limitation . Embodiments herein can be used independently 

or can be combined . [ 0173 ] All ranges disclosed herein are inclusive of the 

endpoints , and the endpoints are independently combinable 

with each other . The ranges are continuous and thus contain 

every value and subset thereof in the range . Unless other wise stated or contextually inapplicable , all percentages , when expressing a quantity , are weight percentages . The 

suffix ( s ) as used herein is intended to include both the singular and the plural of the term that it modifies , thereby including at least one of that term ( e.g. , the colorant ( s ) includes at least one colorants ) . Option , optional , or option ally means that the subsequently described event or circum 

stance can or cannot occur , and that the description includes 

instances where the event occurs and instances where it does 

not . As used herein , combination is inclusive of blends , mixtures , alloys , reaction products , collection of elements , and the like . 

[ 0174 ] As used herein , a combination thereof refers to a combination comprising at least one of the named constitu 

ents , components , compounds , or elements , optionally together with one or more of the same class of constituents , components , compounds , or elements . [ 0175 ] All references are incorporated herein by reference . [ 0176 ] The use of the terms “ a , " " an , ” and “ the ” and 

similar referents in the context of describing the invention ( especially in the context of the following claims ) are to be 

construed to cover both the singular and the plural , unless 

otherwise indicated herein or clearly contradicted by con 

text . It can further be noted that the terms first , second , primary , secondary , and the like herein do not denote any order , quantity , or importance , but rather are used to distin 

guish one element from another . It will also be understood 

that , although the terms first , second , etc. are , in some 

instances , used herein to describe various elements , these 

elements should not be limited by these terms . For example , a first current could be termed a second current , and , similarly , a second current could be termed a first current , without departing from the scope of the various described 

embodiments . The first current and the second current are 

both currents , but they are not the same condition unless explicitly stated as such . [ 0177 ] The modifier about used in connection with a 

quantity is inclusive of the stated value and has the meaning dictated by the context ( e.g. , it includes the degree of error associated with measurement of the particular quantity ) . The 

a 
US 2022/0325332 A1 Oct. 13 , 2022 

16 

conjunction or is used to link objects of a list or alternatives and is not disjunctive ; rather the elements can be used 

separately or can be combined together under appropriate circumstances . What is claimed is : 

1. An agile nucleic acid sensor for measuring a biomarker , the agile nucleic acid sensor comprising : 

a DNA switch that contacts and selectively duplexes with 

a chemical analyte comprising the biomarker ; 

an analysis substrate in electrostatic communication with 

the DNA switch that is disposed on the analysis sub 

strate , such that when the chemical analyte duplexes with the DNA switch , the analysis substrate produces a 

biomarker electrical signal ; 

a transduction member in electrical communication with 

the analysis substrate and that receives the biomarker 

electrical signal and produces a transduction signal in 

response to receiving the biomarker electrical signal ; 

a sensor counter electrode in electrical communication 

with and capacitively coupled to the analysis substrate 

and that receives a counter electrode voltage ; 

a sensor reference electrode in electrical communication and capacitively coupled to the analysis substrate and 

that produces a feedback signal based on electrical 

interactions with a composition that is in fluid contact with the feedback signal and the analysis substrate ; and 

a voltage follower in electrical communication with the 

sensor counter electrode and the sensor reference elec 

trode and that receives the feedback signal from the 

sensor reference electrode and produces the counter 

electrode voltage for the sensor counter electrode based 

on the feedback signal . 2. The agile nucleic acid sensor of claim 1 , wherein the 

DNA switch comprises : 

a DNA nanostructure framework disposed on the analysis substrate and comprising a nucleic acid core , a first 

helix strand protruding from the nucleic acid core and 

attached to the analysis substrate , and a second helix 

strand protruding from the nucleic acid core such that 

the second helix strand is hybridized to the first helix 

strand in an absence of a chemical analyte that prefer entially hybridizes to the first helix strand as compared with the second helix strand , and the second helix 

strand dissociates from the first helix strand when the 

first helix strand is in a presence of the chemical analyte ; 

a particle strand hybridized to the second helix strand ; and 

a reporter particle attached to the particle strand and disposed proximate to the analysis substrate when the 

second helix strand is hybridized to the first helix strand in absence of the chemical analyte and that changes the electrical potential of the analysis substrate depending 

on whether the second helix strand is hybridized to the 

first helix strand . 

3. The agile nucleic acid sensor of claim 1 , wherein when 

the chemical analyte is hybridized to the first helix strand : 

the reporter particle remains attached to the nucleic acid 

core , and is sterically or thermodynamically precluded from 

interacting with first helix strand . 4. The agile nucleic acid sensor of claim 1 , wherein the transduction member comprises a transimpedance amplifier . 5. The agile nucleic acid sensor of claim 4 , wherein the 

transduction member further comprises a transistor . 

6. The agile nucleic acid sensor of claim 5 , further comprising a drain voltage source in electrical communica 

tion with the transistor and that produces a drain voltage for 

the transistor . 7. The agile nucleic acid sensor of claim 1 , further comprising a voltage setpoint source in electrical commu 

nication with the voltage follower and that produces the 

counter electrode voltage for the voltage follower . 8. The agile nucleic acid sensor of claim 1 , further comprising a signal processor in electrical communication 

with the transduction member and that receives the trans 

duction signal from the transduction member . 9. The agile nucleic acid sensor of claim 1 , further 

comprising an interface pad in electrical communication the transimpedance amplifier . 10. The agile nucleic acid sensor of claim 1 , further comprising a substrate on which the DNA switch , the 

analysis substrate , the transduction member , the sensor 

counter electrode , the sensor reference electrode , and the voltage follower are disposed . 11. An agile nucleic acid sensor array comprising a 

plurality of agile nucleic acid sensors arranged in array . 12. The agile nucleic acid sensor of claim 11 , further comprising a signal processor , wherein , for each agile nucleic acid sensor , the signal 

processor is in electrical communication with a trans 

duction member , and the signal processor receives a 

transduction signal from the transduction member . 13. The agile nucleic acid sensor of claim 11 , further comprising a plurality of interface pads , such that each 

transimpedance amplifier is in electrical communication 

with an interface pad . 14. The agile nucleic acid sensor of claim 11 , further comprising a substrate on which the agile nucleic acid 

sensors are disposed . 15. The agile nucleic acid sensor of claim 11 , wherein the agile nucleic acid sensors individually comprise : 

a DNA switch that contacts and selectively duplexes with 

a chemical analyte comprising the biomarker ; 

an analysis substrate in electrostatic communication with 

the DNA switch that is disposed on the analysis sub 

strate , such that when the chemical analyte duplexes with the DNA switch , the analysis substrate produces a 

biomarker electrical signal ; 

a transduction member in electrical communication with 

the analysis substrate and that receives the biomarker 

electrical signal and produces a transduction signal in 

response to receiving the biomarker electrical signal ; 

a sensor counter electrode in electrical communication 

with and capacitively coupled to the analysis substrate 

and that receives a counter electrode voltage ; 

a sensor reference electrode in electrical communication and capacitively coupled to the analysis substrate and 

that produces a feedback signal based on electrical 

interactions with a composition that is in fluid contact with the feedback signal and the analysis substrate ; and 

a voltage follower in electrical communication with the 

sensor counter electrode and the sensor reference elec 

trode and that receives the feedback signal from the 

sensor reference electrode and produces the counter 

electrode voltage for the sensor counter electrode based 

on the feedback signal . 16. The agile nucleic acid sensor of claim 15 , wherein the 

DNA switch comprises : 
US 2022/0325332 A1 Oct. 13 , 2022 

17 

a DNA nanostructure framework disposed on the analysis substrate and comprising a nucleic acid core , a first helix strand protruding from the nucleic acid core and 

attached to the analysis substrate , and a second helix 

strand protruding from the nucleic acid core such that 

the second helix strand is hybridized to the first helix 

strand in an absence of a chemical analyte that prefer entially hybridizes to the first helix strand as compared with the second helix strand , and the second helix 

strand dissociates from the first helix strand when the 

first helix strand is in a presence of the chemical analyte ; 

a particle strand hybridized to the second helix strand ; and 

a reporter particle attached to the particle strand and disposed proximate to the analysis substrate when the 

second helix strand is hybridized to the first helix strand 

in absence of the chemical analyte and that changes the electrical potential of the analysis substrate depending 

on whether the second helix strand is hybridized to the 

first helix strand . 17. The agile nucleic acid sensor of claim 16 , wherein 

when the chemical analyte is hybridized to the first helix 

strand : the reporter particle remains attached to the nucleic acid core , and is sterically or thermodynamically precluded from interacting with first helix strand . 18. A process for measuring a biomarker with an agile nucleic acid sensor array , the process comprising : 

operating the agile nucleic acid sensor array that com prises : 

a plurality of agile nucleic acid sensors arranged in 

array and that individually comprise : 

a DNA switch ; 

an analysis substrate in electrostatic communication 

with the DNA switch that is disposed on the 

analysis substrate ; 

a transduction member in electrical communication 

with the analysis substrate ; 

a sensor counter electrode in electrical communica 

tion with and capacitively coupled to the analysis 

substrate ; 

a sensor reference electrode in electrical communi 

cation and capacitively coupled to the analysis 

substrate ; and 

a voltage follower in electrical communication with 

the sensor counter electrode and the sensor refer 

ence electrode ; 

for individual agile nucleic acid sensor in the agile nucleic 

acid sensor array : 

producing a counter electrode voltage by the voltage follower ; 

subjecting the sensor counter electrode to the counter 

electrode voltage from the voltage follower ; 

contacting the DNA switch with a chemical analyte comprising the biomarker , producing , by the analysis substrate , a biomarker elec 

trical signal in response to the chemical analyte contacting the DNA switch ; 

receiving , by the transduction member , the biomarker 

electrical signal from the analysis substrate and 

producing a transduction signal from the biomarker electrical signal ; determining the impedance of the analysis substrate 

from the transduction signal ; 

estimating kinetic rate constants for each contact 

between the chemical analyte and the DNA switch ; 

and 

combining the kinetic rate constants and producing a 

mean kinetic rate constant with uncertainty quanti fication for the kinetic rate constants ; 

aggregating the mean kinetic rate constants for the plu rality of agile nucleic acid sensor in the agile nucleic 

acid sensor array ; and 

producing a kinetic fingerprint for the chemical analyte 

from the mean kinetic rate constants . 

19. The process of claim 18 , further comprising : 

reading the kinetic fingerprint from the agile nucleic acid 

sensor array ; 

providing the kinetic fingerprint as input to a machine 

learning model for classification ; 

providing the kinetic fingerprint as input to an Al - en 

hanced simulator ; updating training data for the Al - enhanced simulator 

machine learning models ; 

providing a physical model of the agile nucleic acid 

sensor and kinetic fingerprint measurement to the AI 

enhanced simulator machine learning models ; combining the kinetic fingerprint , physical models , and 

parameter uncertainties using machine learning model 

to generate physics enhanced training data with uncer tainty quantification ; and 

combining Al - enhanced simulator training data with the 

sensor array kinetic fingerprint by analyte classification 

machine learning to produce analyte classification with uncertatinty quantification .