U.S. Patent Aug. 22, 2023 Sheet 18 of 20 US 11,733,191 B2

FIG. 18


U.S. Patent Aug. 22, 2023 Sheet 12 of 20

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ame HEET apenioop
oo nFETpp
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US 11,733,191 B2


US 11,733,191 B2

3

about the threshold voltage (V;) of 0 V resulted in a highly
non-linear pH response (inset). (c) Constant current mode
‘operation of the FET was performed by using a proportional-
integral-derivative (PID) controller to monitor I,, and con-
tinually adjust the PID voltage (Vpz,)- Vpyp Was summed
with V,,; prior to being applied to the gate contact. (4) The
change in Vpyp as a function of measuring standard pH
buffer solutions ranging from pH 4 to pH 10 using a glass pH
microelectrode. The PID set-point was set to a value of I, to
allow device operation either at 2,24: OF at the threshold
voltage of 0 V;

FIG. 8 shows electrical characterization of dual-gated
monolayer MoS, FET for biological sensing applications.
(a) Measurement schematic for characterizing a dual-gated
2DFET for remote biosensing. The MoS, 2D semi-conduct-
ing channel spans the source (S) and drain (D) contacts.
While the source contact is grounded, a constant potential
(Vp) is applied to the drain contact driving a current across
the 2D channel. Channel conduction is
controlled by a voltage applied to the silicon substrate,
which forms the global back-gate (B) or to the metal
top-gate (G). (b) Top view optical image of an array of 2D
MoS, FETs. (c) Transfer characteristics of a dual-gated 2D
FET showing drain current (1) as function of the top-gate
voltage (Vg) while stepping back-gate voltage (Vz). (d)
The change in Vz. as function of top-gate threshold voltage
(V, rq) is shown. A linear regression to the data (n=5) is used
to determine the signal amplification (@) of Vg at the
back-gate. The error bars report the standard error defined as
the standard deviation of the population mean;

FIG. 9 shows electrical calibration and pH sensitivity
measurements of dual-gate 2D field-effect transistors
(dg2DFETs) when operated in a constant current mode. (a)
Schematic representation of constant current mode opera-
tion of dg2DFETs.* A proportional-integral-derivative (PID)
controller was used to maintain the channel current (I,,) at a
constant value. Control of I, was achieved by continually
adjusting the back-gate voltage (Vc) in response to
changes in the top-gate potential (V,,,,) applied either using
a waveform generator or from the output of a pH sensor
(V,,_). A DC offset voltage (V,,) was summed with V,,. 10
determine the optimal operation region of the dg2DFET. (b)
The time-variant response of Vz under PID control is
shown when the top-gate is biased with a 1 Hz AC sine wave
signal with a peak-to-peak amplitude of 200 mV. (c)
Response of V,,¢, when measuring standard buffer solutions
from pH 4 to 10. The error bars represent the expanded
uncertainty (k=2) of the measurement. (inset) Time-series

data, relative to a reference potential value, show the s

response of V;,¢, when measuring standard buffer solutions
from pH 4 to 10;

FIG. 10 shows a comparison of pH sensitivity and reso-
lution between a commercially sourced n-channel silicon

field-effect transistor (nFET) and a 2D dual-gated MoS, 5

transistor (dg2DFET) operating under closed-loop propor-
tional-integral-derivative (PID) control when measured
using a glass microelectrode. The error bars in all cases
represent the expanded uncertainty (k=2) of the measured
quantity shown on the y-axis of the plot. (a) The change in
the nFET channel current (AI,,) when operating the device
in open-loop, as a function of phosphate buffered saline
(PBS) solutions adjusted to different pH values. (b) The
change in the PID control voltage (AV p,p) as a function of
solution pH when operating the nFET devices with PID
control. (c) The change in AV pp as a function of solution
pH when operating dg2DFETs under PID control. (insets)

electrostatically 2

&

&

4
Underlying time-series data from nFETs and dg2DFETs that
were analyzed to obtain the plots in panels (a), (b) and (c);

FIG. 11 shows pH resolution of phosphate buffered saline
solutions measured at a bandwidth of 10 Hz;

FIG. 12 shows (a) a comparison of the pH resolution
(ApH) as a function of pH when operating the n-channel
silicon field-effect transistor (nFET) in open-loop, the nFET
under PID control and the 2D dual-gated MoS, transistor
(dg2DFET) under PID control. (b) Power spectral density
(PSD) of the channel current, I,,, of nFETs under open-loop
operation and under PID control. (c) Power spectral density
(PSD) of the channel current, I,, of dg2DFETs s under
open-loop operation and under PID control;

FIG. 13 shows measurements of the activity of the proline
directed kinase, Cdk5 and the effect of the custom designed.
therapeutic polypeptide, p5, on modulating its activity. (a)
(top) The molecular structure of the pathological Cdk5/p25
complex when phosphorylating a substrate protein, histone
HI, during adenosine triphosphate (ATP) hydrolysis. (bot-
tom) The reaction scheme of Cdk5-mediated phosphory-
lation of serine or threonine residues in histone. Upon
hydrolysis of ATP, a single proton is released causing a slight
acidification of the surrounding medium. (b) The change in

5 the measured gate voltage (AVp,p) of a n-channel silicon

field-effect transistor (nFET) upon Cdk5-mediated phospho-
rylation as a function of the substrate protein, histone H1,
concentration ((H1]). (c) Molecular representation of p5
interactions with the CdkS/p25 complex that result in a
decrease in its activity. (d) AV pp as function of p5 concen-
tration ([p5]) shows decreasing Cdk5 activity. The concen-
tration of Cdk5/p25 and histone H1 were held a fixed value
for each measurement in the plot. The error bars in (b) and
(d) represent the expanded uncertainty (k=2) in AVp,p:

FIG. 14 shows steady-state measurements of CdkS activ-
ity using dual-gated field-effect transistors (dgFETs). The
change in the solution pH was used as a reporter to detect
and quantify enzyme-mediated phosphorylation of the pro-
tein histone H1. The change in the back-gate threshold
(Vy5¢) as a function of histone H1 concentration ({H1])
showed a monotonic increase. A simple model was used to
estimate the activity coefficient, k,=(9.1+0.9) wM;

FIG. 15 shows activity of CdkS/p25 that was determined
using a radioactively labeled adenosine triphosphate (y-?2P-
ATP) assay as a function of the concentration of the protein
histone ({H1]). A model was used to determine the activity
coefficient, k,=(12.1#2.3) uM. The error bars represent the
standard deviation in Cdk5 activity;

FIG. 16 shows a time-series measurements of enzyme
catalyzed phosphorylation of histone HI were measured
with a dual-gated field-effect transistor (dgFET). The histone
concentrations ([H1]) were 9.1 1M, 12.7 uM, 18.2 wM and
a control sample with no histone. The solid lines depict a
first order kinetics model that describes the time course of
phosphorylation, while the dashed lines represent an esti-
mate of the reaction velocity during the first 100 s after a
change in the signal was detected;

FIG. 17 shows a functional diagram of a remote biosens-
ing configurations using silicon field-effect transistors;

FIG. 18 shows a functional diagram of a remote biosens-
ing configuration with an isolation element;

FIG. 19 shows a functional diagram of closed-loop feed-
back of ion-sensitive field-effect transistors (ISFETs) or
other chemical field-effect transistors (chemFETs); and

FIG. 20 shows a lock-in detection scheme to improve the
signal-to-noise (SNR) ratio of biochemical sensing,



U.S. Patent Aug. 22, 2023 Sheet 3 of 20 US 11,733,191 B2

fe) fe) 203, 205 203, 205 214 |
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209 218
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FIG. 3


U.S. Patent Aug. 22, 2023 Sheet 19 of 20 US 11,733,191 B2

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FIG. 19


US 11,733,191 B2

9

204 in composition 208, analyte sensor 202; changing, in
response to the electrical perturbation, voltage signal 203
from target voltage 205; receiving, by reference sensor 207,
feedback control signal 209 from feedback controller 210;
actively nulling, by reference sensor 207, the difference
between the voltage signal 203 and the target voltage 205
when analyte sensor 202 is electrically perturbed by analyte
204 to maintain analyte sensor 202 at target voltage 205 for
suppression of electrical noise fluctuations in closed-loop
controlled chemical apparatus 200 that affect voltage signal
203; receiving, by gate terminal 212 of transistor 211, gate
potential 214 such that drain current 215 of transistor 211 is
maintained at the constant value in response to receipt of
feedback control signal 209 by reference sensor 207,
wherein transistor 211 is operated at an optimal transduction
condition comprising peak sensitivity of transistor 211 and
minimum noise of transistor 211 based on reference sensor
207; receiving, by feedback controller 210, transduction
signal 216; determining setpoint 217 from transfer charac-

teristics of transistor 211; determining, by feedback control- 2

ler 210, the deviation of transduction signal 216 from
setpoint 217; producing, by feedback controller 210, feed-
back control signal 209 that minimizes the deviation of
transduction signal 216 from setpoint 217 based on control

model 218; and communicating feedback control signal 209 2:

from feedback controller 210 to reference sensor 207 and
suppressing the electrical noise fluctuations to perform
closed-loop control of closed-loop controlled chemical
apparatus 200.

The process also can include receiving, by input terminal
219 of phase-sensitive detector 213 that is in electrical
communication with drain terminal 221 of transistor 211,
drain current 215 from drain terminal 221; communicating,
by output terminal 220 of phase-sensitive detector 213 that
is in electrical communication with source terminal 222 of
transistor 211, oscillating voltage 223 to source terminal
222; comparing, by phase-sensitive detector 213, drain
current 215 to oscillating voltage 223; producing, by phase-
sensitive detector 213, direct current signal 224 that is
proportional to a phase difference between drain current 215
and oscillating voltage 223; and communicating direct cur-
rent signal 224 to feedback controller 210 as transduction
signal 216.

The process can include receiving, by summer 225 in
electrical communication with analyte sensor 202, feedback
controller 210, and gate terminal 212, feedback control
signal 209 from feedback controller 210; receiving, by
summer 225, voltage signal 203 from analyte sensor 202;
summing, by summer 225, feedback control signal 209 and

voltage signal 203 to produce gate potential 214; and s

communicating gate potential 214 from summer 225 to gate
terminal 212.

Closed-loop controlled chemical apparatus 200 and pro-
cesses disclosed herein have numerous beneficial uses,

including linearizing the response of closed-loop controller 5

chemical apparatus 200, maintaining the transducer 229 at
its optimal operating point for high-resolution measure-
ments, tuning the dynamic range of the measurement in
some embodiments, and actively suppressing the measure-
ment noise using the feedback controller 210. Advanta-
geously, closed-loop controlled chemical apparatus 200
overcomes limitations of technical deficiencies of conven-
tional compositions such as highly nonlinear sensor
response, high flicker noise at measurement bandwidths of
interest, and non-uniform measurement resolution across the
sensor dynamic range. Further, closed-loop chemical appa-
ratus 200 overcomes limitations of conventional composi-

&

10

tions through the composition of the compound sensor,
transducer and feedback controller.

Closed-loop controlled chemical apparatus 200 and pro-
cesses herein unexpectedly transforms the sensor response
to provide consistently low noise operation across all of the
dynamic range, provides a linear sensor response, and
drastically improves measurement resolution through active
noise suppression. Moreover, closed-loop controlled chemi-
cal apparatus 200 provides integrated sensors for chemical
sensing of liquid or gas to sense analytes of interest.

The articles and processes herein are illustrated further by
the following Example, which is non-limiting.

EXAMPLE

Rapid, quantitative therapeutic screening for Alzheimer’s
enzymes enabled by optimal signal transduction with tran-
sistors.

Field-effect transistors (FETs) have a long history as
sensitive and label-free bioanalytical tools. Since that time
FETs have been adapted for numerous applications ranging
from measurements of protein-ligand interactions, monitor-
ing of ocean acidification, low-cost DNA sequencing,
enzyme measurements, and the detection of ionic action
potentials in nerve and other neuronal systems. FET sensors
are fabricated using nanomanufacturing processes that lever-
age silicon-based complementary metal-oxide semi-conduc-
tor (CMOS) technology. More recently, the emergence of 2D
semi-conducting materials has resulted in new FET-based
chemical sensors, and novel device geometries such as
dual-gate FETs, which provide ~100-fold higher sensitivity
than silicon devices, while simultaneously improving the
signal-to-noise ratio (SNR) of the measurements.

The sensitivity and resolution of pH measurements are
metrics of device function and are used, in particular, to
validate the performance of new FET structures and designs.
Conventional ISFET technology has been optimized to
return a pH sensitivity that approaches the Nernst potential
of 59.5 mV per unit change in pH at room temperature.
Efforts to improve the sensitivity of pH measurements have
led to the exploration of dual-gate FETs, which leverage the
asymmetric capacitive coupling between the top- and back-
gate with the device channel to amplify small pH signals.
Using this approach, recent measurements have demon-
strated the amplification of pH signals by 2-fold with silicon
devices and 37-fold (2.25 V/pH) to 75-fold (4.4 V/pH)
higher than the Nernst value using novel channel materials
and gate structures. However, the improvement in sensitivity
does not always result in better pH resolution for silicon
devices. As a result, the pH resolution of silicon ISFETs
range from 50x10~* for commercial ion sensitive field-effect
transistors (ISFETs) to as low as 10x10-* for silicon nanow-
ire devices. On the other hand, dual-gate FETs fabricated
with a 2D MoS, channel and with a room temperature ionic
liquid top-gate dielectric have been demonstrated with a pH
resolution as small as 92x10-° at a bandwidth of 10 Hz. In
particular, the devices, which were operated in a low-noise
regime of the atomically thin channel, exhibit a linear
scaling in pH resolution improvement with device gain.

In order to drive adoption of high-resolution FET mea-
surements within bioanalytical applications, we show how
techniques developed for dual-gate FETs can be applied to
commercially sourced silicon FETs. The techniques allow
silicon devices to achieve pH readout resolution that exceeds
most ISFET results and is on par with a solid-state version
of recently published dual-gate 2D FETs (dg2DFETS).
Improved performance of both nFETs and dg2DFETs was



US 11,733,191 B2

1

CLOSED-LOOP CONTROLLED CHEMICAL
APPARATUS,

CROSS REFERENCE TO RELATED,
APPLICATIONS

‘The application claims priority to U.S. Provisional Patent
Application Ser. No. 62/904,141 filed Sep. 23, 2019, the
disclosure of which is incorporated herein by reference in its
entirety.

STATEMENT REGARDING FEDERALLY,
SPONSORED RESEARCH

This invention was made with United States Government
support from the National Institute of Standards and ‘Tech-
nology (NIST), an agency of the United States Department
of Commerce. The Government has certain rights in the
invention, Licensing inquiries may be directed to the Tech-
nology Partnerships Office, NIST, Gaithersburg, Md.,
20899; voice (301) 975-2573; email tpo@nist.gov: refer-
ence NIST Docket Number 19-057USI.

BRIEF DESCRIPTION

Disclosed is a closed-loop controlled chemical apparatus
comprising: a compound sensor comprising: an analyte
sensor and that: produces, by the analyte sensor, a voltage
signal that varies in response to an analyte that electrically
perturbs the analyte sensor, such that an electrical perturba-
tion changes the voltage signal from a target voltage, the
voltage signal referenced to a reference voltage provided by
a reference sensor through a composition comprising the
analyte; the reference sensor in electrical communication
with the analyte sensor through the composition and that:
receives a feedback control signal from a feedback control-
ler; and actively nulls the difference between the voltage
signal and the target voltage when the analyte sensor is
perturbed by the analyte to maintain the analyte sensor at the
target voltage for suppression of electrical noise fluctuations
in the closed-loop controlled chemical apparatus that affect
the voltage signal; a transistor comprising a gate terminal
that is in electrical communication with the analyte sensor,
such that: the gate terminal receives a gate potential such
that a drain current of the transistor is maintained at a
constant value in response to receipt of the feedback control
signal by the reference sensor, such that the transistor is
operated at an optimal transduction condition comprising
peak sensitivity of the transistor and minimum noise of the

transistor based on the reference sensor; the feedback con- 5

troller in electrical communication with the transistor and
that: receives a transduction signal; determining a deviation
of the transduction signal from a setpoint, the setpoint
determined by transfer characteristics of the transistor; pro-

duces the feedback control signal that minimizes the devia-

tion of the transduction signal from the based on a control
model; and communicates the feedback control signal to the
reference sensor for suppression of electrical noise fluctua-
tions in the closed-loop controlled chemical apparatus.
Disclosed is a process for performing closed-loop control
of a closed-loop controlled chemical apparatus, the process
comprising: producing, by the analyte sensor, the target
voltage; contacting the analyte sensor and the reference
sensor with the composition; electrically perturbing, by the
analyte in the composition, the analyte sensor; changing, in
response to the electrical perturbation, the voltage signal
from the target voltage; receiving, by the reference sensor,

&

&

2

the feedback control signal from the feedback controller;
actively nulling the difference between the voltage signal
and the target voltage when the analyte sensor is electrically
perturbed by the analyte to maintain the analyte sensor at the
target voltage for suppression of electrical noise fluctuations
in the closed-loop controlled chemical apparatus that affect
the voltage signal; receiving, by the gate terminal of the
transistor, the gate potential such that the drain current of the
transistor is maintained at the constant value in response to
receipt of the feedback control signal by the reference
sensor, wherein the is operated at an optimal transduction
condition comprising peak sensitivity of the transducer and
minimum noise of the transistor based on the feedback
control signal; receiving, by the feedback controller, the
transduction signal; determining the setpoint from transfer
characteristics of the transistor; determining, by the feed-
back controller, the deviation of the transduction signal from
the setpoint; producing, by the feedback controller, the
feedback control signal that minimizes the deviation of the
transduction signal from the setpoint based on the control
model; and communicating the feedback control signal from
the feedback controller to the reference sensor and suppress-
ing the electrical noise fluctuations to perform closed-loop
control of the closed-loop controlled chemical apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS,

The following description should not be considered lim-
iting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a closed-loop controlled chemical appara-
tus;

FIG. 2 shows a closed-loop controlled chemical appara-
tus;

FIG. 3 shows a closed-loop controlled chemical appara-

S3

FIG. 4 shows a closed-loop controlled chemical appara-
tus;

FIG. 5 shows a readout 230 for a closed-loop controlled
chemical apparatus;

FIG. 6 shows an electrical characterization of an n-chan-
nel field-effect transistor (nFET) for biosensing. (a) left:
Schematic of the electrical characterization setup of the
nFET. A constant voltage. V;p, is applied to the drain contact
while the source is grounded. The transfer characteristics of
the device are obtained by sweeping the top-gate voltage,
V,,, to electrostatically control the channel current (I)).
right: An image of the packaged nFET used in this work. (b)
The transfer curve of the nFET is shown by measuring I, as
a function of a sweep of Vg. (inset) The device transcon-
ductance as a function of Vg. The voltage at peak transcon-
ductance determines the point of maximum sensitivity and is
used to optimally bias the device for biosensing:

FIG. 7 shows calibration of pH using a commercially
sourced n-channel silicon field-effect transistor (nFET). The
error bars in all cases represent the expanded uncertainty
(k=2) of the measurand. (a) The nFET was operated in
open-loop by directly measuring the changes in the drain
current (I,,) due to changes in the gate potential (Vz¢)
applied to the top-gate contact (G). A constant offset poten-
tial (V,) was summed with the potential (V,,,,) generated by
a glass pH microelectrode prior to being applied to the gate
contact. (b) The change in I,, as a function of measuring
standard pH buffer solutions ranging from pH 4 to pH 10
using a glass pH microelectrode. Setting V, to operate the
device at peak transconductance (2, ,.,4) resulted in a linear
response over the measured pH range. Operating the device



U.S. Patent Aug. 22, 2023 Sheet 16 of 20 US 11,733,191 B2

dgFET é
0.03

rs 0.02
a
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Pe

ob
0 425 250 375
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FIG. 16


U.S. Patent Aug. 22, 2023 Sheet 8 of 20 US 11,733,191 B2

a

Ves (V}



US 11,733,191 B2

21

8. The closed-loop controlled chemical apparatus of claim
7, wherein the protein comprises an enzyme implicated in
Alzheimer’s disease.

9. The closed-loop controlled chemical apparatus of claim
1, wherein the analyte comprises a therapeutic agent that
restores a nonpathological function of a pathogenic form of
a protein.

10. The closed-loop controlled chemical apparatus of
claim 1, wherein the closed-loop controlled chemical appa-
ratus has a pH resolution of 3.5x107* pH units that is more
than three times greater than a pH resolution of an ion-
sensitive field-effect transistor operated in absence of the
feedback control signal.

11. A process for performing closed-loop control of the
closed-loop controlled chemical apparatus of claim 1, the
process comprising:

producing, by the analyte sensor, the target voltage;

contacting the analyte sensor and the reference sensor

with the composition;

electrically perturbing, by the analyte in the composition,

the analyte sensor;
changing, in response to the electrical perturbation, the
voltage signal from the target voltage;

receiving, by the reference sensor, the feedback control

signal from the feedback controller;

actively nulling, by the reference sensor, the voltage

signal when the analyte sensor is electrically perturbed
by the analyte to maintain the analyte sensor at the
target voltage for suppression of electrical noise fluc-
tuations in the closed-loop controlled chemical appa-
ratus that affect the voltage signal;

receiving, by the gate terminal of the transistor, the gate

potential such that the drain current of the transistor is
maintained at the constant value in response to receipt
of the feedback control signal by the reference sensor,
wherein the transistor is operated at an optimal trans-
duction condition comprising peak sensitivity of the
transistor and minimum noise of the transistor based on.
the reference sensor;

receiving, by the feedback controller, the transduction

signal;

determining the setpoint from transfer characteristics of

the transistor,

determining, by the feedback controller, the deviation of

the transduction signal from the setpoint;

producing, by the feedback controller, the feedback con-

trol signal that minimizes the deviation of the trans-
duction signal from the setpoint based on the control
model; and

communicating the feedback control signal from the 5

feedback controller to the reference sensor and sup-

22
pressing the electrical noise fluctuations to perform
closed-loop control of the closed-loop controlled
chemical apparatus.
12. The process of claim 1, wherein the transduction

5 signal comprises the drain current from the transistor.

4

5

0

0

13. The process of claim 11, further comprising:
receiving, by an input terminal of a phase-sensitive detec-
tor that is in electrical communication with a drain
terminal of the transistor, the drain current from the
drain terminal;
communicating, by an output terminal of the phase-
sensitive detector that is in electrical communication
with a source terminal of the transistor, an oscillating
voltage to the source terminal;

comparing, by the phase-sensitive detector, the drain

current to the oscillating voltage;

producing, by the phase-sensitive detector, a direct cur-

rent signal that is proportional to a phase difference
between the drain current and the oscillating voltage;
and

communicating the direct current signal to the feedback

controller as the transduction signal.

14. The process of claim 11, wherein the gate potential
comprises the voltage signal from the analyte sensor.

15. The process of claim 11, further comprising:

receiving, by a summer in electrical communication with

the analyte sensor, the feedback controller, and the gate
terminal, the feedback control signal from the feedback
controller;

receiving, by the summer, the voltage signal from the

analyte sensor;

summing, by the summer, the feedback control signal and

the voltage signal to produce the gate potential; and
communicating the gate potential from the summer to the
gate terminal.

16. The process of claim 11, wherein the trans
prises a field-effect transistor.

17. The process of claim 11, wherein the analyte com-
prises a pathological form of a protein.

18. The process of claim 17, wherein the protein com-
prises an enzyme implicated in Alzheimer’s disease.

19. The process of claim 11, wherein the analyte com-
prises a therapeutic agent that restores a nonpathological
function of a pathogenic form of a protein.

20. The process of claim 11, wherein the closed-loop
controlled chemical apparatus has a pH resolution that is at
least three times greater than a pH resolution of an ion-
sensitive field-effect transistor operated in absence of the
feedback control signal.

istor com-

+e ee


US 11,733,191 B2

15

The measurements were repeated when operating the
nFET under PID control as seen from FIG. 8c. The time-
series of the PID controller output (FIG. 106, inset) were
analyzed identically to the open-loop data to yield a pH
sensitivity, 8V,</8pH=58.7 mV (R?=0.988) as seen from
FIG. 106, consistent with the expected value of Vieysr at
room temperature. The error bars at each measured pl
value, which represent the expanded uncertainty (k=2) in the
PID output voltage, are a direct measure of ApH. We found
that, on average, ApH=(7.2+0.3)x10TM at a bandwidth of 10
Hz or an improved over nFETs operating in open-loop.

Both modes of nFET operation described above were
compared with pH measurements performed using
dg2DFETs. FIG. 10c (inset) shows time-series measure-
ments of pH sensitivity of PBS buffers using dg2DFETs
when they were operated using PID control as described
previously in FIG. 8a. An analysis of the pH time-series (see
Experimental section for details) yielded sensitivity, 8V ;¢/

dpH-384 mV (R?-0,999) as seen from FIG. 10c, which >

represents an ~6.5-fold amplification Of Vyyjy.- at room
temperature. The error bars in the figure estimate the
expanded uncertainty (k=2) of the AV; at each pH value.
This in turn allowed the estimation of the pH resolution
using the expression APH=AV 5o/OXV Nemnsi)=(3.940.7)x
10-* at a bandwidth of 10 Hz, or ~2-fold better than the
nFET devices operating under PID control.

As seen from FIG, 12a and FIG. 11, the pH resolution of
nFETs can be substantially improved by operating them
under PID control. Under this mode of operation, the low
intrinsic noise and the high channel currents of the nFETs
allow their performance to approach that of custom-built
dg2DFETs (FIG. 12a). To better understand the improve-
ment in nFET performance under PID control, we measured
the power spectral density (PSD) of the channel current
noise, Sz, as seen in FIG. 126. Under open-loop operation
(FIG. 125), the devices exhibit 1/f noise scaling as observed
by others in the literature.'**° The root mean squared
(RMS) noise in the channel current (81,5) was then deter-
mined using the expression VJ,,,8,,df to be 2.5 nA in the
open-loop case for a bandwidth of 10 Hz. The PID controller
greatly suppresses 1/f noise as seen from FIG. 126, pink.
When operating under PID control, we found 81, to be 0.18
nA, or an order of magnitude lower for a bandwidth of 10
Hz, and directly results in the improved ApH seen in FIG.
12a.

An improvement in the channel current noise is also
observed for the dg2DFETs when operating under PID

control, in comparison with the open-loop case, as seen from 5

FIG. 12c. At a bandwidth of 10 Hz, 81, decreased from 7.0
pA for open-loop operation to 2.1 pA when operating under
PID control. However, in contrast to the nFETs, the dual-
gate devices have an intrinsic gain of ~6.5, which improves

their overall performance. In order to directly compare the

nFETs with dg2DFETs when both devices were operating
under PID control, we determined the relative error for each
case as 8Ip/Ip, where the channel current, I;, is also the
current set-point for the PID controller. For the nFETs this
value was 40.5 j1A to coincide with g,,,.,..4. and 50 nA for the
dg2DFETs. This results in d1,/1,~4.3x10-° for the nFETs,
and 81,/1,-4.2x10° for the dg2DFETs. The order of mag-
nitude lower relative noise in the nFETSs is offset by the gain,
, resulting from the dual-gated structure of the dg2DFETs
as measured in FIG. 7 and is consistent with the measured
improvement in ApH as seen in FIG. 12a (nFETp,p; and
dg2DFET),

&

&

16

While the pH resolution of the nFETs can approach that
of the dg2DFETs with a moderate internal gain, their per-
formance is expected to fall short of high gain devices such
as ionic liquid gated dual-gate FETs. The highly asymmetric
gate geometries of the ionic liquid gated FETs allows the
realization of a>150 while operating in a low-noise regime
similar to the dg2DFETs. The combination of high gain and
low noise allows those devices to resolve pH values as small
as 92x10°°, which is an order of magnitude below the
resolution attainable by the dg2DFETs (FIG. 12a). While
this improved resolution is useful in certain bioanalytical
applications, we show below that the improvements in the
operation modes of nFETs demonstrated here can be lever-
aged to measure both enzymatic activity and the effect of
therapeutics on enzyme function at physiological concen-
trations.

With regard to Cdk5-p25 pathological activity and neu-
rodegeneration, cyclin-dependent kinase 5 (Cdk5) is
involved in neuronal development, memory, and pain sig-
naling. Its physiological activators, the proteins p35 and
p39, trigger the Cdk5-mediated phosphorylation of neuronal
proteins and organelles that are essential for the normal
function of the human nervous system. Factors, which
include environment, lifestyle and genetics, result in an

5 increased uptake of intracellular Ca' that activates the pro-

tease calpain, truncating p35 into the fragments p10 and p25.
The latter is a pathological activator of Cdk5, leading to its
hyperactivation. Multiple cascading effects within the cell
cycle can be traced back to the hyperactivation of CdkS
resulting in the formation of B-amyloid plaques and intrac-
ellular neurofibrillary tangles—the well-known indicators of
neurodegenerative diseases such as Alzheimer’s disease
(AD).

Therapeutic approaches targeting Cdk5-related patholo-
gies have focused on inhibitors such as Aminothizole and
Roscovitine, which bind to the ATP-docking pocket and
prevent CdkS-mediated hyperphosphorylation. However,
targeting the ATP-binding pocket also causes non-specific
interactions with other ATP-mediated cellular reactions,
often causing serious side-effects. This has led to the pursuit
of alternative approaches, such as the use of cholinesterase
inhibitors or antioxidants, which have thus far not resulted
in safe therapeutics.

In past work, we have shown a novel approach to inhibit
the Cdk5-p25 pathology, for example using the 24 amino
acid, p5, obtained through the repeated truncation of p35.
Importantly, these polypeptides act as selective inhibitors of
CdkS pathological hyperactivity in both in vivo and in vitro
experiments. A variant derived from p5, TFP5, which was
designed to cross the blood-brain barrier showed a drastic
decrease in pathology by allowing the rescue of cortical
neurons in transgenic SXFAD AD model mice in vivo.

We used computer simulations to determine the molecular
basis of p5-based inhibition mechanisms—a step towards
developing safe therapeutics for the regulation of Cdk5/p25
hyperactivity. This in turn could also lead to new molecules
that are more selective and therefore safer. The FET-based
measurements developed here can play a central role in this
development cycle by enabling the rapid testing of candidate
molecules. As a first step towards this goal, we demonstrate
the ability of nFETs to measure the activity of Cdk5/p25 and
the effect of pS on re-regulation of this enzyme under
physiological conditions.

Enzymatic Activity of the Pathological Cdk5-P25 Com-
plex. FIG. 13a shows a molecular representation of the
Cdk5-mediated phosphorylation (top) and the phosphory-
lation reaction scheme (bottom). In the presence of an



U.S. Patent Aug. 22, 2023 Sheet 5 of 20 US 11,733,191 B2

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wy

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U.S. Patent Aug. 22, 2023 Sheet 4 of 20 US 11,733,191 B2

200
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FIG. 4


U.S. Patent Aug. 22, 2023 Sheet 17 of 20 US 11,733,191 B2

sensor transducer

prevestnanernsnnnerenasnnenenanneeers “ peensssavevcnnanewew vannuvennnnocesvennnnuernnan 7

FIG. 17


U.S. Patent Aug. 22, 2023 Sheet 7 of 20 US 11,733,191 B2

a b 20 i
a “te,
2 42
— 10 4 on |
ES OT eT HO
i 2
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ne V,=0V
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~AVpip (MV)
2

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# current
SP preampliier ~ 200k 4 i 1 1
4 6 7 9 10
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FIG. 7


US 11,733,191 B2

5
DETAILED DESCRIPTION.

A detailed description of one or more embodiments is
presented herein by way of exemplification and not limita-
tion.

It has been discovered that a closed-loop controlled
chemical apparatus and processes herein provide readout
elements for a biosensor to achieve about a three-fold
improvement in pH resolution over conventional ion-sensi-
tive field-effect transistors (ISFETs). The closed-loop con-
trolled chemical apparatus is operated under closed-loop
control, actively nulling any input to a gate terminal of a
transistor from charge fluctuations at a sensing surface of an
analyte sensor. The improved pH resolution was realized
while as the closed-loop controlled chemical apparatus were
operated in a remote configuration with a pH sensing surface
off-chip and connected electrically to a gate terminal of a
transistor. The closed-loop controlled chemical apparatus
has a sensitivity and resolution that measured activity
pathological form of kinase Cdk5, an enzyme implicated in
Alzheimer’s disease, and demonstrated effectiveness of a
polypeptide, p5, as a therapeutic agent in restoring non-
pathologic function of CdkS. Accordingly, the closed-loop
controlled chemical apparatus and processes herein provide
drug discovery and clinical diagnostics.

In an embodiment, with reference to FIG. 1, FIG. 2, FIG.
3, and FIG. 4, closed-loop controlled chemical apparatus
200 includes: compound sensor 201 including: analyte sen-
sor 202 and that: produces, by analyte sensor 202, voltage
signal 203 that varies in response to analyte 204 that
electrically perturbs analyte sensor 202, such that an elec-
trical perturbation changes voltage signal 203 from target
voltage 205, voltage signal 203 referenced to reference
voltage 206 provided by reference sensor 207 through
composition 208 comprising analyte 204; reference sensor
207 in electrical communication with analyte sensor 202
through composition 208 and that: receives feedback control
signal 209 from feedback controller 210; and actively nulls
the difference between the voltage signal 203 and the target
voltage 205 when analyte sensor 202 is perturbed by analyte
204 to maintain analyte sensor 202 at target voltage 205 for
suppression of electrical noise fluctuations in closed-loop
controlled chemical apparatus 200 that affect voltage signal
203; transistor 211 including gate terminal 212 that is in
electrical communication with analyte sensor 202, such that:
gate terminal 212 receives gate potential 214 such that drain
current 215 of transistor 211 is maintained at a constant
value in response to receipt of feedback control signal 209

by reference sensor 207, such that transistor 211 is operated 5

at an optimal transduction condition including peak sensi-
tivity of transistor 211 and minimum noise of transistor 211
based on feedback control signal 209; feedback controller
210 in electrical communication with transistor 211 and that:

receives transduction signal 216; determines a deviation of

transduction signal 216 from setpoint 217, setpoint 217
determined by transfer characteristics of transistor 211;
produces feedback control signal 209 that minimizes the
deviation of transduction signal 216 from setpoint 217 based
on control model 218; and communicates feedback control
signal 209 to reference sensor 207 for suppression of elec-
trical noise fluctuations in closed-loop controlled chemical
apparatus 200.

‘Advantageously, by using feedback control signal 209 to
control reference sensor 207 in combination with analyte
sensor 202 and transistor 211, closed-loop controlled chemi-
cal apparatus 200 has a pH resolution of 3.5x10~> pH units

of a 2

&

&

6

that is more than three times greater than a pH resolution of
an ion-sensitive field-effect transistor operated in absence of
feedback control signal 209.

In an embodiment, with reference to FIG. 1, transduction
signal 216 includes drain current 215 from transistor 211.

According to an embodiment, with reference to FIG. 2
and FIG. 4, closed-loop controlled chemical apparatus 200
includes phase-sensitive detector 213 (e.g., a lock-in ampli-
fier) in electrical communication with transistor 211 such
that: input terminal 219 of phase-sensitive detector 213 is in
electrical communication with drain terminal 221 of tran-
sistor 211 and that receives drain current 215 from drain
terminal 221; and output terminal 220 of phase-sensitive
detector 213 is in electrical communication with source
terminal 222 of transistor 211 and that communicates oscil-
lating voltage 223 to source terminal 222, such that phase-
sensitive detector 213: compares drain current 215 to oscil-
lating voltage 223 and produces direct current signal 224
that is proportional to a phase difference between drain
current 215 and oscillating voltage 223; and communicates
direct current signal 224 to feedback controller 210 as
transduction signal 216.

In an embodiment, with reference to FIG. 1 and FIG. 2.
gate potential 214 includes voltage signal 203 from analyte
sensor 202.

In an embodiment, with reference to FIG. 3 and FIG. 4,
closed-loop controlled chemical apparatus 200 includes
summer 225 in electrical communication with analyte sensor
202, feedback controller 210, and gate terminal 212 and that:
receives feedback control signal 209 from feedback control-
ler 210; receives voltage signal 203 from analyte sensor 202;
and sums feedback control signal 209 and voltage signal 203
to produce gate potential 214; and communicates gate
potential 214 to gate terminal 212

Reference sensor 207 and analyte sensor 202 are inde-
pendent electrodes that can be electrically conductive or
capacitively coupled to be in electrical communication with
other elements. A size of reference sensor 207 and analyte
sensor 202 can be selected so that compound sensor 201 can
be disposed in a suitable container for receipt of composition
208 that can be communicated from composition source
231. Composition source 231 can be a macrofluidic or
microfluidic flow system or static reservoir. Suitable pumps
and delivery conduits (c.g., tubing) can fluidically connect
composition source 231 to compound sensor 201 for deliv-
ery of composition 208 to compound sensor 201. Flow
between composition source 231 and compound sensor 201
can be controlled via devices such as flow meters.

Composition 208 can include analyte 204 as well as other
constituents such as solvents, other compounds (e.g., small
molecule drugs), excipients, cells, proteins, and the like.
‘Analyte 204 can be any item that electrically perturbs
analyte sensor 202 by presence of analyte 204 at analyte
sensor 202, including direct contact therebetween or indirect
contact such as interaction through an electrical double layer
proximate to a surface of analyte sensor 202, wherein the
electrical perturbation can occur by a formal electrical
charge of analyte 204 or higher order electrical moment of
analyte 204 such as a dipole interaction, quadrupole inter-
action, and the like effecting the electrical perturbation of
analyte sensor 202. Exemplary analytes 204 include small
molecule drugs, proteins, cells, polymers, inorganic com-
pounds, and the like. A concentration of analyte 204 in
composition 208 can be selected as an unknown value of
known value, e.g., for calibration of compound sensor 201.
In an embodiment, analyte 204 includes a pathological form
of a protein. It is contemplated that the protein can be an



United States Patent

331

US011733191B:

(12) (0) Patent No.: US 11,733,191 B2
Balijepalli et al. (45) Date of Patent: Aug. 22, 2023
(54) CLOSED-LOOP CONTROLLED CHEMICAL —_(58)__ Field of Classification Search
APPARATUS cPc GOIN 27/4145; GOIN 33/54373; BOLL
3/502715; BOIL 2200/027
(71) Applicant: Government of the United States of See application file for complete search history.
America, as represented by the
Secretary of Commerce, Gaithersburg, (56) References Cited
MD (US)
ws) U.S. PATENT DOCUMENTS
(72) Inventors: Arvind Kumar Balijepalli, 4879517 A * 11/1989 Conne GoIN 27/4148
Washington, DC (US); Curt Andrew a emery ‘24/15
Richter, Olney, MD (US); Son Truong 10,385,377 B2 8/2019 Takechi et al.
Le, Germantown, MD (US) (Continued)
(73) Assignee: GOVERNMENT OF THE UNITED ~
STATES OF AMERICA, AS OTHER PUBLICATIONS
REPRESENTED BY THE Zafar, S., et al., “Silicon Nanowire Field Effect Transistor Sensors
SECRETARY OF COMMERCE, with Minimal Sensor-to-Sensor Variations and Enhanced Sensing
Gaithersburg, MD (US) ACS Nano, 2018, p. 6577-6587, vol. 12.
(*) Notice: Subject to any disclaimer, the term of this (Continued)
pase vento be 308 adinsted under 35 primary Examiner — Jermele M Hollington
fo DSO) OY AOS CONS: Assistant Examiner — Temilade § Rhodes-Vivour
(21) Appl. No.: 17/029,999 (74) Attorney, Agent, or Firm — Office of Chief Counsel
for National Institute of Standards and Technology
(22) Filed: Sep. 23, 2020
(57) ABSTRACT
(65) Prior Publication Data A closed-loop controlled chemical apparatus includes: a
US 2021/0088463 Al Mar. 25, 2021 compound sensor including: an analyte sensor and that:
produces, by the analyte sensor, a voltage signal; a reference
sensor in electrical communication with the analyte sensor;
Related U.S. Application Data a transistor including a gate terminal such that a drain
. . . current of the transistor is maintained at a constant value and
(60) Provisional application No. 62/904,141, filed on Sep. operated at an optimal transduction condition of peak sen-
23, 2019. sitivity and minimum noise of the transistor; a feedback
; controller in electrical communication with the transistor
(51) Int. Cl. and that: receives a transduction signal; determining a
GOIN 27/12 (2006.01) deviation of the transduction signal from a setpoint, the
GOIN 33/483 (2006.01) setpoint determined by transfer characteristics of the tran-
(Continued) sistor; produces the feedback control signal that minimizes
(52) U.S. Ch the deviation of the transduction signal from the based on a
cPc GOIN 27/12 (2013.01); A6IB 5/14546 control model; and communicates the feedback control

(2013.01); A61B 5/4088 (2013.01);
(Continued)

signal to the reference sensor for suppression of electrical
(Continued)

i
Be



U.S. Patent Aug. 22, 2023 Sheet 10 of 20 US 11,733,191 B2

4.0 pA/DH

AVpip (mV)

A¥ew (nV)

20/0 FET

"

oF 384 mV/pH
~50

~ 100

¥

Avec (mV)

150

~200

T

i
67 68 69 7.0 7.4
pH

FIG. 10


U.S. Patent Aug. 22, 2023 Sheet 9 of 20 US 11,733,191 B2

a oe
preamplifier
PID
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ag 8 Y,
i Oo eee ee: ee
axa v7 (E>. out
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~900



US 11,733,191 B2

19

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.

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 otherwise
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). “Optional” or “optionally” means that the
subsequently described event or circumstance 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, reac-
tion products, and the like.

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.

All references are incorporated herein by reference.

The use of the terms “a” and “an” and “the” and similar
referents in the context of describing the invention (espe-
cially 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. “Or” means “and/or.” It should 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 distinguish one element from another. 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 conjune-
tion “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 circum-
stances.

What is claimed is:
1. A closed-loop controlled chemical apparatus compris-
ing:
‘a compound sensor comprising:
an analyte sensor and that:
produces, by the analyte sensor, a voltage signal that
varies in response to an analyte that electrically

perturbs the analyte sensor, such that an electrical

perturbation changes the voltage signal from a
target voltage, the voltage signal referenced to a
reference voltage provided by a reference sensor
through a composition comprising the analyte:

the reference sensor in electrical communication with

the analyte sensor through the composition and that:

receives a feedback control signal from a feedback
controller;

actively nulls the difference between the voltage
signal and the target voltage when the analyte
sensor is perturbed by the analyte to maintain the
analyte sensor at the target voltage for suppression

10

&

0

50

0

20

of electrical noise fluctuations in the closed-loop
controlled chemical apparatus that affect the volt-
age signal;
a transistor comprising a gate terminal that is in electrical
communication with the analyte sensor, such that:
the gate terminal receives a gate potential such that a
drain current of the transistor is maintained at a
constant value in response to receipt of the feedback
control signal by the reference sensor, such that the
transistor is operated at an optimal transduction
condition comprising peak sensitivity of the transis-
tor and minimum noise of the transistor based on the
feedback control signal;
the feedback controller in electrical communication with
the transistor and that:
receives a transduction signal;
determines a deviation of the transduction signal from
a setpoint, the setpoint determined by transfer char-
acteristics of the transistor;
produces the feedback control signal that minimizes the
deviation of the transduction signal from the setpoint
based on a control model; and
communicates the feedback control signal to the refer-
ence sensor for suppression of electrical noise fluc-
tuations in the closed-loop controlled chemical appa-
ratus.
2. The closed-loop controlled chemical apparatus of claim
1, wherein the transduction signal comprises the drain
current from the transistor.
3. The closed-loop controlled chemical apparatus of claim
1, further comprising a phase-sensitive detector in electrical
communication with the transistor such that:
an input terminal of the phase-sensitive detector is in
electrical communication with a drain terminal of the
transistor and that receives the drain current from the
drain terminal; and
an output terminal of the phase-sensitive detector is in
electrical communication with a source terminal of the
transistor and that communicates an oscillating voltage
to the source terminal, such that the phase-sensitive
detector:
compares the drain current to the oscillating voltage
and produces a direct current signal that is propor-
tional to a phase difference between the drain current
and the oscillating voltage; and
communicates the direct current signal to the feedback
controller as the transduction signal
4. The closed-loop controlled chemical apparatus of claim
1, wherein the gate potential comprises the voltage signal
from the analyte sensor.
5. The closed-loop controlled chemical apparatus of claim
1, further comprising a summer in electrical communication
with the analyte sensor, the feedback controller, and the gate
terminal and that:
receives the feedback control signal from the feedback
controll
receives the voltage signal from the analyte sensor; and
sums the feedback control signal and the voltage signal to
produce the gate potential; and
communicates the gate potential to the gate terminal
6. The closed-loop controlled chemical apparatus of claim
1, wherein the transistor comprises a field-effect transistor.
7. The closed-loop controlled chemical apparatus of claim
1, wherein the analyte comprises a pathological form of a
protein.



US 11,733,191 B2

17

activator protein (¢.g., the pathological p25), Cdk5 catalyzes
the transfer of a single phosphate group from ATP to a serine
or threonine residue in a substrate protein (e.g., histone H1).
The reaction also releases a single proton, thereby causing
the surrounding medium to become slightly more acidic and
decreasing its pH. When using the nFETs, the change in pH
resulted in a change in Vp, shown in FIG. 134, relative to
a control sample with no enzyme, for concentrations of
histone ({H1}) ranging from 9 1M to 25 tM and is shown in
FIG. 13b. All measurements were performed at a physi-
ological concentration of Cdk5/p25 of 18.5 nM. As
expected, the change in Vp, increased monotonically with
increasing histone concentration. A simple model of the
form

(Ht)
YR +H

where k, is the activity of and y is a scaling constant, was fit
to the data in FIG. 135. The value of k, was then estimated
to be (8.2#1.3) uM when measured with the nFETs. This
value was consistent with k,=(9.1+0.9) uM for measure-
ments performed using dg2DFETs (FIG. 14) under identical
solution conditions, our previous measurements using a
y-?P-ATP assay (k,=12.1+2.3 tM) as seen from FIG. 15,
and with literature values of Cdk5/p25 activity! In each
case, the error bars of the estimated quantity represent the
standard error of the measurement.

We leveraged the high time-resolution of our technique to
measure the kinetics of the Cdk5/p25 enzymatic reaction for
histone concentrations of 9.1 wM, 12.7 uM and 18.2 1M as
seen from FIG. 16. In each case, the reaction was initiated
upon the addition of ATP. After minute, we observed a
distinct change in the voltage signal that was indicative of
histone phosphorylation. A control sample with no Cdk5/
p25 showed no change in signal upon the addition of ATP.
A nonlinear regression of the form B(1-e), where f is a
scaling constant, was used to estimate the enzyme rate
constant k, to be (0.35+0.1) min”', consistent with literature
values.

With regard to inhibition of Cdk5/p25 activity with p5,
upon the addition of the 24 amino acid polypeptide, p35. we
observed a strong reduction in Cdk5/p25 activity. A plau-
sible structure of the complex formed between pS and
Cdk5/p25 is shown in FIG. 13c. However, the molecular and
regulatory mechanism of p5 action on Cdk5/p25 activity is
still under investigation. As in FIG. 135, the measurements

were performed with a Cdk5/p25 concentration of 18.5 nM_ 5

and a histone concentration of 25.4 iM. FIG. 13d shows the
change in Vp, when the p5 concentration ([p5]) was
increased from 0.25 iM to 1.2 WM. From the figure, we can
clearly see the effect of p5-based inhibition. Furthermore,

the measurements agree with previous results of the inter-

action of p5 with the Cdk5/p25 complex obtained using a
y-P-ATP assay. In particular, the sharp decrease in Cdk5/
ast [p5]=0.7 uM. This decrease is indicative of
a specific threshold for p5 inhibition and will be studied
further in future work.

We show that the operation of commercially sourced
nFETs can be optimized to achieve a pH resolution of
(7.220.3)x10-*, =3-fold better than traditional ISFETs, and
on par with solid-state dg2DFETs with an intrinsic gain a=8.
Furthermore, the improved performance is attained when the
devices are operated in a remote configuration, with the pH
sensing element located off-chip and connected electrically

3

&

at

18

to the FET. This is in contrast to conventional ISFETs which
use integrated pH sensing membranes that form the gate
dielectric. Our design greatly increases the versatility of
nFETs, allowing them to be rapidly and easily interfaced
with different biochemical sensors. The improved perfor-
mance of the nFETs, operated in a remote configuration, was
shown to be adequate to measure the activity of a patho-
logical form of the proline directed kinase, Cdk5, which has
been implicated to cause numerous neurodegenerative con-
ditions including Alzheimer’s disease. We further con-
firmed, using nFET measurements, the effectiveness of a
custom polypeptide, pS, in re-regulating Cdk5 function.
Together the measurements demonstrate performance of
sensitive bioanalytical measurements using commercially
available FETs.

Remote measurements include a sensor (e.g., pH sensing
element) physically separated from the charge transduction
element but connected electrically that improves system
stability but can suffer from signal degradation due to stray
parasitic interference. The setup in FIG. 17 overcomes some
of the limitations of remote sensor configurations, for
example when the sensor is not in close proximity to the
transducer. In these cases, we place an ultra-low noise

5 voltage follower (F1) with an input impedance that exceeds

100 GQ, in close proximity with the sensing element to
minimize charge degradation along the wire connecting the
sensor to the transducer. The signal from the sensor is
connected to a switch (SI) that is used to isolate the
transducer from the sensor. In one version, the sensor signal
(AV,) is summed with the output of the PID controller
using an adder (A1) and then applied to the FET gate. In a
second version, the PID output is used to bias the reference
electrode (Ref). The difference in voltage between the
reference and pH electrodes is then applied to the FET gate.
The PID operation to maintain the FET in a constant current
mode is identical to the setup described in FIG. 7.

For integrated sensor and readout systems such as ion
sensitive field-effect transistors (ISFETs) or chemical field-
effect transistors (chemFETs), closed loop control can be
used to improve measurement resolution using the setup
seen in FIG. 18. In this case, a voltage applied to the
reference electrode (ref) will change the current in the
channel (I,)) spanning the source (S) and drain (D) elec-
trodes. The output of the PID controller is used to continu-
ally adjust voltage applied to the reference electrode
immersed in the solution bath to maintain a constant I,, in
response to chemical species adsorbing to the sensing sur-
face.

Measurement sensitivity can be improved by orders of
magnitude by performing an AC measurement using phase
sensitive detection (PSD) embedded within the measure-
ment. The modifications to the transducer setup to achieve
this are shown in FIG, 19. As shown in the figure, a reference
AC voltage with a known frequency (F4<) and amplitude
(V,¢) is added to V,,. The applied AC voltage results in an
AC component in Ip. Unlike the DC configuration in FIG.
17, the output of the current preamplifier is input to a
lock-in detector. The input signal is demodulated using two
independent demodulators each operating using a reference
signal separated by a phase angle of 90 degrees. This
two-phase approach returns a demodulated DC signal that
equals R=1,,xG, where G is the gain of the current pream-
plifier. The output of the lock-in amplifier is then connected
to the PID input as described in FIG. 7. The lock-in detection,
scheme shown in FIG. 19 can be implemented without
modification for the system in FIG. 18.



U.S. Patent Aug. 22, 2023 Sheet 6 of 20 US 11,733,191 B2

a sinsnnsinnsni



U.S. Patent Aug. 22, 2023 Sheet 20 of 20 US 11,733,191 B2

[oe

ae
l preamplifier pu]
| : ee * cast | tej eal
Ea

. }

FIG. 20


US 11,733,191 B2

11

achieved in a remote sensing configuration where the pH
sensitive surface is located off-chip and connected electri-
cally to the FETs. The advantage of the remote sensing
approach is that the signal transduction is separated from
sensing, allowing reuse of the electronic components and
minimizing parasitic noise sources. This setup differs from
ISFET studies where the gate dielectric also serves as the pH
sensing membrane.

This Example shows high-resolution pH measurements
by comparing closed-loop transduction using an nFET to the
‘open-loop operation of the same device. The improved
performance establishes that this approach can allow use of
transistors for laboratory grade bioanalytical measurements.
Furthermore, the closed-loop transduction approach can be
applied to a wide range of sensor technologies that could be
based on other stand-alone transistors such as junction FETs
(JFETs) and bipolar junction transistors (BJTs) and even to
integrated sensors such as ISFETs. This readout approach
improves performance of sensor systems based on any of
these transistors. The technique is demonstrated through
measurements of the activity and the effect of customized
polypeptide therapeutics on the kinase Cdk5, which is
implicated Alzheimer’s disease and numerous other debili-
tating disorders.

With regard to n-channel silicon field-effect transistors,
silicon field-effect transistors (nFETs) were soldered onto a
printed circuit board (PCB) prior to measurements using a
commercial probe station. Electrical characterization of the
nFETs was performed using a semiconductor parameter
analyzer. Time-series measurements with the nFETs were
performed similar to the dg2DFET measurements as
described below.

With regard to 2D dual-gate transistor fabrication, mono-
layer MoS, was first transferred onto an oxidized Si sub-
strate (SiO,) with a thickness of 70 nm using the gold-
mediated exfoliation technique. The thickness of the
transferred material was confirmed with Raman spectros-
copy. Optical lithography was used to first pattern the source
(S) and drain (D) contacts followed by electron-beam metal
deposition (80 nm Au on 2 nm Ti) and lift-off in acetone. We
then used a second optical lithography step to define and
etch a 5 jumxS yum channel for each FET. The devices were
then annealed under forming gas (5% H2, 95% Ar) for 24
hours to minimize organic contamination and improve the
contact resistance. This step was followed by the atomic
layer deposition (ALD) of a 20 nm top-gate (TG) Al,O;
dielectric. Finally, another optical lithography step was used
to pattern the top-gate metal, followed by electron-beam
metal deposition (100 nm Au on 10 nm Ti) and lift-off in
acetone.

With regard to remote biological activity measurements,
the enzyme and pH calibration measurements using the
dg2DFETs and nFETs were performed by connecting a pH
sensor to the top-gate metal contact using a shielded coaxial

cable. This allowed electronic components to be separated

from the biological components and thereby reused. In the
present work, a glass combination microelectrode capable of
measurement volumes as small as 50 tL was used as the pH
sensor, although the techniques described here are compat-
ible with other sensing and bioanalytical surfaces that can be
electrically connected to the top-gate metal contact.

With regard to time-series field-effect transistor measure-
ments and PID control, time-si its were
performed by operating the nFETs and dg2DFETs under
proportional-integral-derivative (PID) control. The channel
current, I, was maintained at a constant value by continu-
ously varying the back-gate voltage (V,;,;) in the case of the

&

&

12
dg2DFETs (FIG. 8a) or by adding the controller output to
the signal from the pH sensor (V,,,,) for the nFETs (FIG. 9c)
in response to changes in the top-gate potential.

The PID control system was implemented by first ampli-
fying the channel current, I, using a current preamplifier
(DLPCA-200; FEMTO, Berlin, Germany) using a gain of
either 10° V/A (dg2DFET) or 10° V/A (nFET). The output
of the current preamplifier was then filtered using a 4-pole
Bessel filter with a cutoff frequency of 5 kHz and sampled
with a frequency of 25 kHz using a 14-bit analog-to-digital
converter. The digital PID controller (K,-553.5 mV,
K=9.22x10° s~! and K,=10.4 us) was operated with a
bandwidth of | kHz to maintain the channel current set-
point. Because most biological processes are slow and do
not require high bandwidth measurements, the controller
output was further filtered using a low-pass filter with a
cutoff frequency of 10 Hz prior to being recorded.

With regard to sensitivity and resolution of pH measure-
ments, the pH sensitivity and resolution were established,
wherein a histogram from the raw Vp, time-series data was
computed for each measured pH. A sum of two Gaussian
distribution functions was then fit to the histograms to obtain
the peak positions and standard deviations of the reference
potential and the measured pH signal. The difference in the

5 peak positions between the pH and reference potentials

(AV zp) Was used to determine the pH sensitivity of the
device. The measurement uncertainty (op,)) was then
obtained by propagating the error when determining AV...
For the nFETs, the pH resolution, APH=(kxOp:)/V yermse iS
reported with expanded uncertainty (k=2), where Viera, is
the Nernst potential at room temperature. For the
dg2DFETs, ApH=kopip/(@XV vemss)s Where @. is the device
gain.

With regard to kinase measurement reagents, the activity
of Cdk5/p25 was estimated by measuring the phosphory-
lation of histone H1. All measurements were performed with
18.5 nM of CdkS/p25 in 1x kinase buffer to match physi-
ological conditions using a volume of 50 tL. The substrate
protein histone HI was suspended in deionized water at a
stock concentration of 2 mg/mL and further diluted as
described in the in the Results section. Substrate phospho-
rylation was initiated with a mixture of dithiothreitol (DTT)
and adenosine triphosphate (ATP) with final concentrations
of 250 uM and 5 mM respectively. The measurements were
buffered using 5x kinase buffer, prepared by suspending 25
mM f-glycerol, 50 mM MgCl, 5 mM EGTA, 2.4 mM
EDTA, 1.25 mM MOPS in deionized water (DIW) and
diluting further to form 1x kinase buffer.

We electrically characterized nFET and dg2DFET devices
to evaluate their performance and to determine the optimal
operating conditions for biosensing applications. The
devices were calibrated with standard pH buffer solutions to
determine the gain, a, of the dg2DFET, noise performance,
sensitivity, and resolution. Finally, both device types were
used to measure the activity of the kinase Cdk5, an enzyme
implicated in Alzheimer’s disease and to evaluate the effec-
tiveness of a custom polypeptide, p5, therapeutic agent
in modulating Cdk5 function.

Silicon n-Channel FET Performance. The nFETs (FIG.
6a; right) were first characterized electrically using the
configuration shown in the schematic in FIG. 6a (left). The
transfer characteristics of the device were determined by
recording the drain current (I,,) as a function of the gate
potential (Vg), while the drain voltage (V,) was held
constant. FIG. 66 shows the typical transfer characteristics
(Ip-Vg) for the nFETs. The device exhibits up to orders of
magnitude change in I,, when switching from an off-state to



U.S. Patent Aug. 22, 2023 Sheet 2 of 20 US 11,733,191 B2

200
231
+—~ 208
208 | 298
y io) 222 2
Woy, % 2
Ov oO 293 208 214
Ks
?
207 206 202 212
re) e)
fo)
oo fe)
223] 2157]
220 219
223 215
228
213
24-4]
216] %
217
N 210
209 218
226

FIG. 2


U.S. Patent Aug. 22, 2023 Sheet 13 of 20 US 11,733,191 B2

~AVeip (mV)

a O-P-0 0 6 10 18 20 28
OH ATP ADP {HS} GaN)

Serithr — CAKSI 25

a

~AVpip (MV)

a L

i
00 03 806 O8 12

[pS] (uM)

FIG. 13


U.S. Patent Aug. 22, 2023 Sheet 15 of 20 US 11,733,191 B2

oO

ie

Cdk5 Activity (x10° CPM)
KO ob.

a

0 5 10 15 20 25

[H1] (uM)

FIG. 15


U.S. Patent Aug. 22, 2023 Sheet 1 of 20 US 11,733,191 B2

200
231
hr 208
298 | 2M 229
v
Wl 6 O45 2UL
6°
ie} 203, 205 2 214
es
2 2 2
207 2 202 212
of 295 8
oe) >| 222 fod} 221
OY oO

2154
216—| i
¥
217
5 210
209 218
226

FIG. |


US 11,733,191 B2

7

enzyme implicated in Alzheimer’s disease, such as CDKS.
In an embodiment, analyte 204 includes a therapeutic agent
that restores a nonpathological function of a pathogenic
form of a protein.

‘Analyte sensor 202 produces voltage signal 203 that
initially can be at target voltage 205 in an absence of analyte
204 electrically perturbing analyte sensor 202, and changes
from target voltage 205 in a presence of analyte 204 that
electrically perturbs analyte sensor 202. Target voltage 205
referenced to reference voltage 206 can be a selected value
such as the voltage value of reference voltage 206. When
analyte sensor 202 is electrically perturbed by analyte 204,
the electrical potential of analyte sensor 202 changes from
target voltage 205 to voltage signal 203 so that a larger
potential difference occurs relative to reference voltage 206.
Accordingly, a potential difference between voltage signal
203 and reference voltage 206 is greater than the potential
difference between target voltage 205 and reference voltage
206, wherein the magnitude of the potential difference

between voltage signal 203 and reference voltage 206 2

increases as the strength of the electrical perturbation of
analyte sensor 202 by analyte 204 increases.

With reference to, e.g., FIG. 1, transducer 229 receive
gate potential 214 can be voltage signal 203 or target voltage
205, depending on whether analyte 204 electrically perturbs
analyte sensor 202. Transducer 229 includes optional isola-
tor 232 that can be, e.g., a switch to isolate transistor 211
from compound sensor 201. Isolator 232 can be controlled
by an external signal, e.g., a TTL voltage from an external
source that can be programmatically or user set. Transistor
211 can be of various types such a field-effect transistor
(FET, eg., a silicon FET) that includes gate terminal 212,
source terminal 222, and drain terminal 221 in which gate
terminal 212 receives gate potential 214 and an amount of
drain current 215 (e.g., sourced from power source 227 or
from phase-sensitive detector 213) that flows in an active
channel between source terminal 222 and drain terminal 221
is determined by the magnitude of voltage of gate potential
214 received at gate terminal 212. Transducer 229 can
include amplifier 233 that receives drain current 215 from
drain terminal 221 and that amplifies the drain current for
communication to signal processor 226.

Signal processor 226 is in electrical communication with
transducer 229. Signal processor 226 uses setpoint 217 that
is derived a model of the transfer function of transducer 229,
particularly operation of transistor 211. It is contemplated
that Setpoint 217 is set to operate transistor 211 at its
optimum operating point derived from the transfer charac-
teristics of transistor 211. Here, feedback controller 210

(e.g.. a proportion-integration-derivative (PID) controller) s

receives setpoint 217 and control model 218 to determine
feedback control signal 209 from transduction signal 216
that can be drain current 215 from drain terminal 221 of
transistor 211 or direct current signal 224 from phase-

sensitive detector 213. It should be appreciated that control 5

model 218 can have one of many embodiments that approxi-
mate dynamics of the measurement system. Control model
218, in conjunction with feedback controller 210, evaluates
the difference between transduction signal 216 and setpoint
217 to produce feedback control signal 209 that actively
drives transduction signal 216 to setpoint 217

In some embodiments, signal processor 226 includes
phase-sensitive detector 213. Exemplary phase-sensitive
detector 213 include a lock-in amplifier that determines a
phase difference between oscillating voltage 223 and drain
current 215 from output terminal 220 and input terminal
219, respectively. Here, drain current 215 is received from

&

8

transistor 211 by input terminal 219, and oscillating voltage
223 is provided internally and communicated to source
terminal 222 of source terminal 222 via output terminal 220.

Feedback control signal 209 is communicated from feed-
back controller 210 to reference sensor 207 of compound
sensor 201, Reference sensor 207 is used to actively null the
difference between voltage signal 203 and target voltage 205
caused by analyte 204 through electrical perturbation of
analyte sensor 202. In an embodiment, with reference to
FIG. 5, feedback control signal 209 also can be communi-
cated from feedback controller 210 to readout 230 that can
visually display or store analyte information 240. Readout
230 can include filter 234 that filters feedback control signal
209 and produces filtered signal 235 that is communicated to
digitizer 237 that digitizes filtered signal 235 and produces
digitized signal 238. Digitized signal 238 is received by data
processor 239 that produces analyte information 240 from
digitized signal 238. Analyte information 240 can include
properties of the analyte type that include individual or some
combination of properties such as its concentration in solu-
tion determined by the amplitude of the signal, kinetic
information pertaining to its interaction with the sensor that
further allows discrimination of analyte type from other
species in solution, and the like. Display 241 (c.g. a

| computer monitor) can display analyte information 240, e.g..

as a visual image for a user, and data storage 242 (e.g., a
server or other non-transitory storage medium) can store
analyte information 240.

Closed-loop controlled chemical apparatus 200 can be
made in various ways. In an embodiment, a process for
making closed-loop controlled chemical apparatus 200
includes electrically connecting compound sensor 201 to
transducer 229. Voltage signal 203 from compound sensor
201 communicates analyte information to gate terminal 212
of transistor 211. In another embodiment, isolator 232
isolates transducer 229 from compound sensor 201. Voltage
signal 203 modulates drain current 215 of transistor 211 to
generate transduction signal 216. Transduction signal 216 is
connected electrically to feedback controller 210. In another
embodiment, drain current 215 is connected to input termi-
nal 219 of pha: ensitive detector 213. In this embodiment,
oscillating voltage 223 is electrically connected to the source
terminal of transistor 211. Further, drain current 215 is
compared with oscillating voltage 223 using comparator 228
to produce transduction signal 216. Feedback controller 210
is configured to compare transduction signal 216 with set-
point 217. Using control model 218, feedback controller 210
determines feedback control signal 209, Feedback control
signal 209 is electrically communicated to reference sensor
207 within compound sensor 201. In another embodiment,
feedback control signal 209 is summed with voltage signal
203 by summer 225. Feedback control signal 209 represents
the measured output of closed-loop controlled chemical
apparatus 200.

It should be appreciated that in making closed-loop con-
trolled chemical apparatus 200 elements thereof are
arranged with components to be in communication fluid or
electrical communication so that components provide for
fluid flow or communication of electrical signals amongst
the various electrical components.

Closed-loop controlled chemical apparatus 200 has
numerous advantageous and unexpected benefits and uses.
In an embodiment, a process for performing closed-loop
control of closed-loop controlled chemical apparatus 200
includes: producing, by analyte sensor 202, target voltage
205; contacting analyte sensor 202 and reference sensor 207
with composition 208; electrically perturbing, by analyte



US 11,733,191 B2
Page 2

noise fluctuations in the closed-loop controlled chemical
apparatus.

20 Claims, 20 Drawing Sheets

(51) Int. CL.

AGIB 5/145 (2006.01)
AGIK 38/16 (2006.01)
AGIB 5/00 (2006.01)
(52)
AGIB 5/4839 (2013.01); A61B 5/7203
(2013.01); A61K 38/16 (2013.01); GOIN
33/483 (2013.01)
(56) References Cited

U.S. PATENT DOCUMENTS.

2012/0078071 A1* 3/2012 Bohm AGIB 5/14532

600/345,
2016/0238553 A1* 8/2016 Shachar .. GOIN 27/4145
2017/0350882 A1* 12/2017 Lin GOIN 33/54353
201910246959 A1* 8/2019 Ionescu... . AG1B 5/1477

201910369044 A1* 12/2019 Chang GOIN 27/4146

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essential for neurite
*, Genes and Develop-

* cited by examiner


US 11,733,191 B2

13
an on-state with a steep sub-threshold slope of ~100 mV/dec
at ~300 K and gate leakage current (I,) was ~1 pA. FIG. 6
(inset) shows the transconductance (g,,), obtained by taking
the numerical derivative of the transfer curve. The peak
transconductance of the nFETs Grgect was found to be
78.1 US at a voltage (Vg ymax) of 0.385 V.

With regard to pH sensitivity using n-channel silicon
transistors, the pH sensitivity of the nFETs was measured
using commercial standard pH buffer solutions over a range
of 4 to 10. When operating the device in open-loop (FIG.
7a), we found the pH response to be linear when the device
was operated about g,,,¢,4 8 Seen in FIG. 76. This behavior
is expected when operating the device in the linear regime
of the transfer curve (see FIG. 6). Under these conditions,
we found the pH sensitivity, 51p/8pH~6 wA (R?=0.992)
when V,,-0.1 V, yielding a transimpedance gain of 9.9x10°
VIA, assuming Vyoy.¢¢ OF 59.5 mV at room temperature (300
K). The pH response was drastically different, as expected,
when the device was operated about its threshold voltage
(V;-0 V). In this case, the pH response was linear under
acidic conditions (pH<7), when the pH sensor returned a
positive potential, thereby driving the FET into the inversion
regime. On the other hand, under more basic conditions
(pH>7), the sensor potential was negative causing the nFET
to operate in the sub-threshold regime where the current
decreases exponentially at negative gate potential. The net
result is a pH response that is highly non-linear over the
Therefore, it can be advantageous to
n the linear regime, particularly when
operating over a wide pH range. Additionally, the larger I,,
in the linear regime over its value at V; results in lower
relative noise and improved pH resolution as discussed in
greater detail in later sections.

The sensitivity and performance of the commercial nFET
improved considerably when operated under closed-loop
PID control as shown schematically in FIG. 7c. In this
configuration, the controller continually adds a control volt-
age (Vpyp) to Vx, thereby maintaining I, at a constant
value. A key advantage of this approach is that because the
device will always operate at the same point in its transfer
curve (see FIG. 66), its performance remains consistent
across a wide range of measured pH values. This is clearly
seen in FIG. 7d, where the device exhibits a linear pH
response (R?=0.997) both when the PID controller was set
up to hold the nFET at g,, .,.. (FIG. 74) and when the device
was operating at its V;-of 0 V (FIG. 7d). Furthermore, the pH
sensitivity of the system in both cases, obtained from a linear
regression of the curves in FIG. 8d, was found to be ~56 mV.
per unit change in pH, approaching the Nernst value of 59.5

mV per unit change in pH at room temperature. Finally, the 5

separated gate configuration of the system setup allows the
PID output to be summed with the sensor signal, allows the
controller to operate at higher bandwidths and enables better
suppression of noise, thereby improving pH resolution.
Dual-Gate 2D
formance of the nFETs with the dg2DFETs that we fabri-
cated using atomically thin MoS, films. An optical image of
a representative dg2DFET device is shown in FIG. 8a
(right). The dg2DFETs were electrically characterized fol-
lowing fabrication using the setup shown schematically in
FIG. 8a (left). The transfer characteristics of the device were
measured by recording the drain current (I,,) as a function of
ate potential (V,,) with the drain voltage (V,,) held
1 measurements were repeated for different Vig
to determine the signal amplification (a) due to the asym-
metric capacitance of the top and back gates (FIG. 86).* The
devices exhibited a dynamic range of up to orders of

Performance. We compared the per- 5

&

&

14

magnitude in I,, and a subthreshold slope consistent with
expected behavior for a 20 nm high-k gate dielectric.

For each curve in FIG. 86, the top-gate threshold voltage
(V,,6) was determined from a linear extrapolation of the
peak transconductance to the x-axis. FIG. 7e plots the
back-gate voltage (V,,;) against the top-gate threshold volt-
age (V,7<). This allowed the determination of the device
gain using the expression a=dV ,4/dV,7¢- The value of a
for devices measured as part of this work was then deter-
mined numerically from a linear regression to the data in
FIG. 6c, resulting in a=8+1, or times larger than conven-
tional dual-gate silicon devices. The measured value of «. is
in good agreement with theoretical predictions for devices
with a 20 nm Al,O, top-gate dielectric and 70 nm SiO,
bottom oxide.

The dg2DFETs presented here do not operate in the
quantum capacitance limited regime. This is due to the fact
that, in the inversion regime where the dg2DFETs operate,
the top- and back-gate capacitances (C74~0.4 tF/em? and.
Cyo~0.05 pF/em?) are more than an order of magnitude
smaller than the quantum capacitance (Cg~4 1F/cm?) of the
2D channel. This allows us to ignore the effects of Co,
giving rise to the simplified expression for the device gain,
oAWV g6/WV.26-Cro/Cre

With regard to a pH sensitivity of dual-gate 2D FETs, the
dg2DFETs were operated in a constant current mode shown
in FIG. 9a. A PID controller was used to maintain I, at a
preset value by continuously varying Vg in response to
changes in V7. The controller current set-point (50 nA) and
a DC offset voltage applied to the top gate (V,=+0.5 V) were
optimized to operate the device in the linear and low-noise
region of the transconductance curve as determined from
FIG. 8c. The performance of the device was then validated
by applying a sine wave at a frequency of 1 Hz and
peak-to-peak amplitude of 200 mV to the top-gate and
measuring the response of Vc (FIG. 96) as regulated by the
PID controller. The device gain, a, was then obtained from
the ratio of the amplitudes of Vgc, to Vz¢ to yield a=5.820.1,
consistent with the value obtained from the data in FIG. 7c.

The pH sensitivity of the dg2DFETs was measured by
remotely connecting a pH sensor to the top-gate metal
contact using a shielded cable. A switch was used to alter-
natively either ground the top-gate or connect it to the pH
sensor. Time-series measurements of the system response,
under PID control, to commercial standard buffer solutions
from pH 4 to pH 10 are shown in FIG. 9. 4d (inset). The
time-series data were analyzed to yield the pH response
curve in FIG. 9c with a sensitivity, 5V;¢/6pH=236.3 mV
(R2=0.998).

With regard to pH resolution of n-channel silicon and
dual-gate MoS, transistors, pH resolution (0.4 pH) of nFETs
and dg2DFETs at a bandwidth of 10 Hz were determined.
when measuring phosphate buffered saline (PBS) and are
summarized in FIG. 11. Following a procedure in which a
switch alternatively connected the gate terminal to the PBS
solution or to ground. This method allowed the measure-
ments of time-series of nFETs operated in an open-loop as
seen in FIG. 10a. A histogram of the time-series was then
used to determine the mean value of I,, for each measured
pH solution and the expanded uncertainty (k=2). As seen
from FIG. 10a, a linear regression of the measured pH data
yielded a sensitivity, 51,/3pH=4 yA (R2-0.974) when
V,-0.1 V, similar to the value obtained from the data in FIG.
9b. By inverting the curve in FIG. 9b and propagating the
uncertainty in I, we determined ApH with an expanded
uncertainty (k=2) to be (22+2)x10-* at a bandwidth of 10 Hz
in the open-loop configuration.



U.S. Patent

pH

NFET open-top
nFETow
dg2DFET

Aug. 22, 2023 Sheet 11 of 20

6.72
177
6.0

35

6.87 6.88
27.4 24.0
6.6 8.0
26 5.8

FIG. 11

US 11,733,191 B2

6.96 7.05
18.6 25.4
7.3 78
5.0 23


U.S. Patent Aug. 22, 2023 Sheet 14 of 20 US 11,733,191 B2

O.00//  )
0 5 10 15 20
[H1] (um)

FIG. 14