Patent Application: US-201314651538-A

Abstract:
the invention combines recent advances in barcode technology with portable wireless communication devices to engineer a simple and low - cost chip - based multiplex wireless detection system . the system can analyze multiple targets of interest simultaneously in minutes and is applicable to detection of pathogens or contaminants in a wide range of fields including medicine , agriculture and the environment .

Description:
in this specification and in the claims that follow , reference will be made to a number of terms that shall be defined to have the meanings below . all numerical designations , e . g ., dimensions and weight , including ranges , are approximations that typically may be varied (+) or (−) by increments of 0 . 1 , 1 . 0 , or 10 . 0 , as appropriate . all numerical designations may be understood as preceded by the term “ about ”. the singular form “ a ”, “ an ”, and “ the ” includes plural references unless the context clearly dictates otherwise . the term “ comprising ” means any recited elements are necessarily included and other elements may optionally be included . “ consisting essentially of ” means any recited elements are necessarily included , elements that would materially affect the basic and novel characteristics of the listed elements are excluded , and other elements may optionally be included . “ consisting of ” means that all elements other than those listed are excluded . embodiments defined by each of these terms are within the scope of this invention . the term “ ligand ” or “ probe ” as used herein refers to a capture molecule , organic or inorganic , or group of molecules that exhibits selective and / or specific binding to one or more organic or inorganic targets . targets may include specific sites of a receptor , a probe , another molecule ( organic or inorganic ) or target or whole microscopic organisms ( unicellular or multicelluar ) such as a pathogen . there can exist more than one ligand for a given target . the ligands may differ from one another in their binding affinities for the target . examples of ligands include nucleotide - based ligands ( aptamers , oligonucleotides , and so forth ), amino acid - based ligands ( antibodies , peptides , proteins , enzymes , receptors and so forth ), polysaccharide - based ligands ( for example hyaluronan ), antigens , hormones , including peptide - hormones , lipid / phospholipid - hormones and monoamine hormones , and any other molecule capable of binding to an organic or inorganic target . multiplex may be understood as the ability to detect the presence - of more than one target simultaneously . the multiplex detection system may include barcodes , metal , semiconductor , or organic based nanostructures or molecules , ( e . g . organic dyes ). barcodes may include any type of structure or system that allows a target to be distinguished . barcodes that may be used with the present invention include magnetic , optical ( i . e . quantum dots , organic dyes ), electrical , dna and lithographic barcodes . as used herein , a “ quantum dot ” ( qd ) is a semiconducting photoluminescent material , as is known in the art ( for example , see alivasatos , science 271 : 933 - 937 ( 1996 )). non - limiting examples of qds include : cds quantum dots , cdse quantum dots , cdse / cds core / shell quantum dots , cdse / zns core / shell quantum dots , cdte quantum dots , pbs quantum dots , and / or pbse quantum dots . as is known to those of skill in the art , cdse / zns means that a zns shell is coated on a cdse core surface ( ie : “ core - shell ” quantum dots ). the shell materials of core - shell qds have a higher bandgap and passivate the core qds surfaces , resulting in higher quantum yield and higher stability and wider applications than core qds . quantum dot barcodes refers to microbeads containing different combinations of fluorescent semiconductor nanocrystals . each microbead may include a unique optical signature that identifies the surface conjugated molecule . approximately 10 , 000 to 40 , 000 different optical barcodes may be engineered using 5 - 6 different color quantum dots and six intensity levels ( 9 ). this enables significant multiplexing and these barcodes can detect targets in a flow cytometer ( 10 - 13 ) or microfluidic channel ( 14 , 15 ) as well as through other means . wireless communication device refers to any device using radio - frequency , infrared , microwave , or other types of electromagnetic or acoustic waves in place of wires , cables , or fibre optics to transmit or receive signals or data , and that the device includes a camera for acquiring images , signals or data and electronic components to sustain analysis of the images , signals or data . wireless communication devices include smart phones , tablets , smart watches , personal assistant devices , and portable computers . the present invention demonstrates that the integration of a multiplex detection system , such as barcodes , with portable wireless communication devices , such as smartphone or tablet technology , may be used in a system for multiplex detection and identification of targets of interest and wireless transmission of data . the detection device contemplates integrating a portable wireless communications device with the multiplex detection system where the optics , excitation source , and detector may be combined into a single device the size of the current smartphone or tablet . with reference to fig1 a , in one embodiment , the system 100 may include a substrate 120 for receiving the multiplex detection system thereon , and a wireless communication device 130 . as illustrated in fig1 a the multiplex detection system may include a primary label 110 , a secondary label 112 and a ligand 111 to a target of interest coupled to the primary label 110 and secondary label 112 . the secondary label may be coupled to the same target - specific ligand as the one bound to the primary label , or to another target - specific ligand . in fig1 , the primary label 110 is represented as quantum dot barcodes . it should be understood that other multiplexing systems may be used and that primary labels other than qd barcodes may be used . the system may also include an excitation source 140 for exciting the primary label 110 and secondary label 112 , an optical means having an objective 150 for collecting the optical emission from the excited primary label and secondary label , one or more filters 142 , 144 for filtering the beams from the excitation source and the emissions from the primary label and secondary label . the system 100 may also include a centralized facility 160 for wirelessly receiving the data collected by the wireless communication device 130 for storing or further analyzing the data collected . examples of excitation sources that may be used with the system of the present invention may include light emitting diodes , laser diodes , lasers , and lamp burners . examples of non - light emitting excitation sources include electrical potential sources . examples of optical filters that may be used with the system of the present invention may include absorbing glass filters , dye filters , color filters , dichroic mirrors , beam splitters , and thin - film polarizers . in one embodiment of the present invention , the wireless communication device itself may include at least one of the excitation source , the objective for collecting the emission from the excited barcodes and secondary labels and one or more filters . the substrate may be any suitable substrate for receiving the multiplex detection system and that can be portable . substrates can be static or dynamic . a static substrate may include a substantially flat surface capable of receiving the multiplex detection system or capable of barcode deposition . substrates may include glass slides , cellulose membranes , paper , plastic membranes or slides and so forth . a dynamic substrate may include a micro - channel or capillary network . for convenience , the static substrate may also include one or more wells that may help organize the multiplex detection system . the wells may also serve to hold the multiplex system on the substrate . in the case of a static substrate the multiplex detection system may be coupled to a surface of the substrate . in the case of a dynamic substrate , the multiplex detection system may flow in solution through the substrate . with reference to fig1 a , toward the development of such a system 100 for multiplex detection and / or identification and wireless data transmission , engineering a multiplex - chip platform , which may be portable , may be manufactured by assembling primary labels , such as barcodes 110 on the surface of a microfabricated substrate 120 , such as a slide . the substrate depicted in fig1 a includes a plurality of indentations or microwells 115 for receiving the primary labels and secondary labels . samples may be added to the substrate 120 , and a wireless communication device 130 may be used to collect or capture signals 125 from the substrate 120 , deconvolve the signals and associate the signals with a specific target , such as a pathogen , disease marker , or contaminant . ligands or probes that are specific to different targets of interest may be conjugated or attached onto a primary label , such as a barcode , and to a secondary label . the conjugation or attachment of a ligand or probe to a barcode or a secondary label will depend on the type of ligand or probe used and the surface chemistry of the barcode and secondary label . examples of conjugation techniques include carbodimmide mediated , maleimide , n - hydrosuccinimide or thiol - metal chemistry , dna - hybridization , antigen - to - antibody , protein - to - small molecule ( streptavidin - to - biotin ). by way of example , oligonucleotide - based ligands or probes may be conjugated onto the surface of each barcode using carbodiimide chemistry . with continued reference to fig1 a , the barcodes 110 with the conjugated ligand 111 may then be arranged onto microfabricated chips or substrates 120 including a plurality of indentations 115 . the indentations may be 3 μm - diameter wells ( micropep ). barcodes beads may be prepared by using any technique known in the art , for example the technique of flow - focusing ( 16 ). the barcode beads may be of any suitable size . for example , they may be of a size similar to the diameter of the microwells . a solution , preferably a buffer solution , including a panel of barcodes may be added directly onto the chip . the barcodes may then be allowed to settle into each well . these microbeads may not easily desorb off of the chip during subsequent assays after they are bound to the microchip . as illustrated in fig4 , the filling efficiency on the chip is determined by the concentration and size of the beads . in our assays , we typically use a filling efficiency of 25 to 50 % to maximize access of the capture molecule to the bead surface . fig1 b is a photograph of a typical microwell chip containing different barcodes in each well . the black arrow illustrates a drop of a sample to be analyzed on the chip . the sample may be allowed to incubate at about room temperature or more . the chip may then be rinsed and imaged using the wireless communication device . fig1 c is a micrograph of the image captured by a wireless communication device of quantum dot barcodes assembled on the surface of a chip . in another embodiment , the system of the present invention may be used in a method for simultaneously detecting multiple targets of interest in a sample . in one embodiment , the method may include : ( a ) contacting the sample with a substrate having a multiplex detection system distributed therein , the multiplex detection system being capable of producing different signals upon interaction with the multiple targets , each signal corresponding to a particular target ; and ( b ) collecting the signals from the substrate with a wireless communication device , and ( c ) analyzing the collected signals using the wireless communications device , whereby the multiple targets in the sample can be simultaneously detected . in one aspect of the present invention the analysis of step ( c ) includes quantifying the multiple targets in the sample . the applicants developed a simple method to assemble a primary label , such as microbead barcodes , on the surface of a chip . in one embodiment , glass slides may be microfabricated with 3 . 0 μm - diameter indentations . a solution of microbead barcodes , which may be about 3 . 0 μm sized microbead , having different combinations of fluorophores may then be added to the chip . the microbead barcodes may settle into each microwell . once bound to the microwell , these microbeads may not desorbed from the surface of the microwell . the microbeads may be held in place by non - covalent forces . the concentration and size of the barcodes may determine the filling efficiency ( see fig4 ). with reference to fig1 a , a sample of interest , such as a subject &# 39 ; s biological fluid or an environmental sample , may be incubated on a chip 120 containing primary label 110 , ligand 111 and secondary label 112 for a suitable amount of time , for example for about 20 minutes , rinsed or washed , and placed on the system for analysis . an excitation source 140 may then be used to excite the primary label and the secondary label . the optical signal may be collected by an objective . the optical signal may be filtered with one or more filters 142 , 144 imaged using a wireless communications device camera 130 , and analyzed using the wireless communications device 130 or remotely in a centralized facility 160 or by other wireless communication devices or both . in one embodiment of the present invention , the collected signal data may be interpreted as positive (+) or negative detection (−) using a custom - designed algorithm which may be integrated / downloaded / uploaded onto the wireless communications device . the data may be sent wirelessly to a centralized facility for further evaluation , storage , or for the mapping and tracking of pathogens or contaminants . detection of a specific target occurs when the ligand binds the target and the overall signal of the microbeads comprises of both the primary label and the secondary label . in one embodiment of the method of the present invention , a primary label may be bound to a first ligand and a secondary label may be bound to a second ligand , both ligands having affinity for the same target of interest . the first and the second ligand may be the same or different . after a suitable incubation time , a washing step may be added after the binding of a label to a ligand to wash away any unbound material . a sample of interest may then be incubated together with both the primary label bound to a target - specific ligand and with the secondary label bound to a target - specific ligand for a suitable incubation time . the incubation may be followed by at least one washing step to remove any unbound material . the washing step may then be followed by the excitation and analysis step . in another embodiment , the secondary label may be added to the primary label bound to a ligand . then a sample of interest may be incubated with the primary label bound to a ligand and with the secondary label for a suitable incubation time . a washing step to remove any unbound material may be performed after adding the sample . the excitation step may be performed after the washing step ( s ). in another embodiment , a sample of interest may be incubated with the primary label bound to a ligand for a suitable amount of incubation time . a secondary label may then be added for a suitable incubation time . a washing step to remove any unbound material may be performed before adding the secondary label , after adding the secondary label or both before and after adding the secondary label . the excitation step may be performed after the washing step ( s ). fig1 b shows a microwell chip in accordance with one embodiment of the present invention , having different barcodes in each well . in a biological assay , the sample , for example about 10 μl , may be disposed or placed on the chip ( see black arrow ), incubated at 40 ° c ., rinsed , and imaged . with reference to fig1 c , a wireless communications device camera may capture the image of the different barcodes assembled on the surface of the chip ( in the case of fig1 c , five different qd barcodes ). these barcodes may be excited a suitable excitation source such as a hg lamp ( λex = 350 / 50 ). the optical signal may be collected by an objective . the optical signal may be filtered , for example with 430 nm long - pass filter , and imaged using a wireless communication device , such as an apple iphone ™ 4s smartphone with an exposure time of 0 . 05 s . fig1 d demonstrates the wireless transmission of the optical image to other wireless communication devices . advantages of the present invention include : ( a ) detection of one or multiple ( i . e . more than one ) targets ( i . e . multiplexing ) as compared to other cellphone - based approaches ; ( b ) the deposition of barcodes on the chip , compared to those stored in solution , enables higher portability of barcodes and reduces the number of steps in the barcode assay process ; ( c ) the device itself would also be portable ( not much bigger than the size of a smartphone or tablet - to which it will be attached ); ( d ) the current detection platforms for identifying quantum dot barcodes require expensive instruments and detectors and would be prohibitive in their use in remote and resource - limited settings and in the field ( 12 , 13 ); ( e ) the systems and methods of the present invention are simple and easy to use because the procedures are few and uncomplicated , thus obviating the need for a skilled technician ; and ( f ) detection is relatively quick ( less than 30 minutes ) from deposition of sample to obtaining results of the analysis . in order to aid in the understanding and preparation of the present invention , the following illustrative , non - limiting examples are provided . in this embodiment , quantum dots ( cdses alloyed - zns capped ) of peak emission wavelength 540 nm (“ qd540 ”) were purchased from cytodiagnostics and used as instructed . quantum dots ( qds ) of peak emission wavelengths 589 nm (“ qd589 ”) and 640 nm (“ qd640 ”) were synthesized and characterized according to published procedures ( 18 - 20 ) and stored in chloroform at room temperature until use . other types of qd nanoparticles may also be used . in this embodiment , qd barcodes were prepared by mixing together the quantum dots ( qd540 , qd589 , and qd640 ) in different ratios with a polymer - based solution . the polymer solution consisted of poly ( styrene - co - maleic anhydride ) ( 32 %, cumene terminated ) from sigma - aldrich dissolved in chloroform , with the polymer concentration at 4 wt %. the resultant quantum dot polymer solution was then introduced into a nozzle system from ingeniatrics using a syringe pump from harvard apparatus at a rate of 0 . 9 ml / hour , as well as double - distilled ( dd ) water as the focusing fluid at a rate of 180 ml / hour . the nozzle system was then submerged inside a beaker partially filled with dd water . the polymeric barcode beads were synthesized in situ , and the beads formed a white colloidal suspension in the water . after synthesis , the valve was closed and the beads were stabilized by overnight stirring and then collected . the beads were filtered using 35 μm bd falcon nylon mesh strainer cap , and characterized using an automated beckman coulter vi - cell counter , and stored in dd water at 4 ° c . until use . the quantum dot concentrations required for preparing the seven different barcodes are presented in table 2 . for high dispersion and microwell filling efficiency of the five barcode beads ( b1 , b3 , b4 , b5 , b6 from fig8 ) on a glass slide having a plurality of 3 μm - diameter wells ( micropep ), samples with concentration 3 × 10 7 bead / ml were prepared for each . then , 2 μl of each sample was mixed with 35 μl of dd water and 5 μl of dd water containing 1 % tween to produce a final mixture concentration of 6 × 10 6 beads / m l . the mixture was then sonicated for 5 minutes to reduce bead aggregation before depositing 30 μl of it on the microwell chip , which was rinsed with dd water and allowed to dry prior to deposition . the chip was then placed in an enclosed drying chamber containing dessicant to prevent dust particle contamination , and then allowed 2 hours to dry before imaging . note that increasing the bead concentration in the mixture increases the microwell fill efficiency ( fig4 ), but with greater potential for aggregation . conjugation of dna capture strands ( i . e . amine groups present on the 5 ′ end of c1 to c7 ) to their corresponding barcode beads ( i . e . carboxylic acid groups present on the polymeric 170 surface of b1 to b7 ) was done through reaction with 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide hydrochloride ( edc ). dna capture strands from bio basic inc ., purchased hplc - purified and used without further purification , were designed with an amine group and 12 base spacers on the 5 ′ end . they were first prepared at a concentration of 10 pmol / μl in te buffer and stored at 4 ° c . until further use . to conjugate , edc was first dissolved in mes buffer ( ph 5 , 100 mm ) at a concentration of 100 mg / ml . approximately 106 beads were mixed with 100 μl of the edc solution , and it was allowed to activate the bead carboxyl groups for 10 minutes . then , 2 . 88 μl of the dna capture strand solution , corresponding to 28 . 8 pmol of dna , was added to the bead solution . the reaction was allowed to take place overnight . to validate the conjugation , 1 μl of dd water containing 5 % tween was added to the 180 bead solution , centrifuged at 3000 g for 5 minutes . then , 50 μl of the supernatant was extracted . the same conjugation procedures described above were performed for the control cases for each barcode ( i . e . no conjugation ), except dd water was added in place of beads . in a black 96 - well plate , 10 μl of the supernatants from all seven conjugation cases , 10 μl of the supernatants from all seven control case , as well as 10 μl of four blank cases containing only dd water , were each added to individual wells . sybr green i from invitrogen , dissolved in dmso , was first diluted to 1 : 10000 dilution by adding 1 μl of it to 10 ml of te buffer , then 190 μl of the dilution was added to each of the sample - containing wells . all reactions were incubated at room temperature for 15 minutes before being read using a plate reader from bmg labtech . amount of conjugation for each barcode was then determined by comparing the fluorescence of the conjugation cases with their respective controls containing no beads . that is , lower signal indicates higher amount of conjugation . results were converted to efficiency in percentages ( see fig7 ). to finish the conjugation process , after the 50 μl of the supernatant was extracted for validation , the remaining supernatant was removed . then , the conjugated beads were washed 195 twice with 100 μl of dd water containing 0 . 05 % tween and centrifuged at 3000 g for 5 minutes to remove any non - conjugated dna capture strands . the conjugated beads were then stored in 100 μl dd water containing 0 . 05 % tween at 4 ° c . until further use . sensitivity assays were performed directly on the microwell chips for all infectious disease dna target strands ( t1 to t5 of fig8 ) and their respective conjugated barcode beads ( b1 - c1 to b5 - c5 of fig8 ). the sensitivity results are illustrated in fig5 . dna target strands from bio basic inc ., purchased hplc - purified and used without further purification , were prepared in increasing concentrations of 0 , 5 , 10 , 50 , 100 , 500 , 1000 , and 2000 fmol / μl in te buffer . dna detection strand from idt dna technologies with alexa647 fluorophore on the 5 ′ end , purchased hplc - purified and used without further purification , were prepared with concentration of 100 pmol / μl in te buffer . both dna target and detection strand samples were stored at 4 ° c . until further use . to perform the assay , 1 μl of the conjugated bead sample , corresponding to approximately 104 conjugated beads , was deposited on a microwell chip for each assay condition and let dry for 1 hour . then , 1 μl of each dna target strand sample was mixed with 5 μl of hybridization buffer ( 10 × ssc , 0 . 1 % sds , heated to 60 ° c . ), 3 μl of dd water , and 1 μl of dna detection strands or dd water ( for the blank condition ). this resulted in a total hybridization volume of 10 μl for each assay condition , which include blank , 0 , 5 , 10 , 50 , 100 , 500 , 1000 , and 2000 fmol . the hybridization solution for each assay condition was deposited over the conjugated bead spots on the microwell chips and 215 incubated at 40 ° c . for 20 minutes . the microwell chips were then submerged in 10 ml of washing buffer ( 0 . 5 × ssc , 0 . 1 % sds , heated to 40 ° c . ), washed by agitation for 20 s , then let dry for 5 minutes before being imaged . note that care must be taken so that the washing buffer does not dry and crystallize over the sample spots . for the 3 - plex multiplexing assay ( fig2 a and b ), 2 μl of each conjugated barcode sample ( b1 - c1 ( green ), b4 - c4 ( yellow ), and b6 - c6 ( red )), corresponding to approximately 2 × 10 4 barcodes each , were mixed together with 18 μl of dd water to produce a 4 × dilution factor of the original . the dilution was to reduce bead aggregation after deposition on chip , which may confound barcode resolution during analysis . to perform the assay , 5 μl of the conjugated barcode mixture , corresponding to approximately 1 . 25 × 10 3 conjugated beads , was deposited on a microwell chip for each assay condition and let dry for 3 hours . then , 2 μl of t1 and t6 ( concentration of 2 pmol / μl each ) was mixed with 40 μl of hybridization buffer ( 10 × ssc , 0 . 1 % sds , heated to 60 ° c . ), 14 μl of dd water , and 16 μl of the detection strand ( concentration of 100 pmol / μl ). this resulted in a total hybridization volume of 70 μl . from this , 10 μl of the hybridization solution was deposited over the conjugated barcode spots on the microwell chip and incubated at 40 ° c . for 20 minutes . the microwell chip was then submerged in 10 ml of washing buffer ( 0 . 5 × ssc , 0 . 1 % sds , heated to 40 ° c . ), washed by agitation for 20 s , washed again in another 10 ml of washing buffer to further reduce non - specific binding , then let dry for 5 minutes before being imaged . note that care must be taken so that the washing buffer does not dry and crystallize over the sample spots . cross reactivity between all five dna target strands ( t1 to t5 ) and their corresponding conjugated barcodes ( b1 - c1 to b5 - 05 ), as well as negative and positive control cases ( b6 - c6 and t6 , and b7 - c7 and t7 , respectively ), was studied ( fig3 a - f ). first , 6 μl of each conjugated barcode sample , corresponding to approximately 6 × 10 4 barcodes each , were mixed together with 126 μl of dd water to produce a 4 × dilution factor of the original . the dilution was to reduce bead aggregation after deposition on chip , which may confound barcode resolution during analysis . to perform the assay , 8 μl of the diluted conjugated barcode mixture , corresponding to approximately 2 × 10 4 conjugated beads , was deposited on a microwell chip for each multiplexing case and let dry for 4 hours . then , 2 μl of each target case ( dd water for the negative conditions , and corresponding dna target strand sample with concentration of 2 pmol / μl for the positive conditions ) was mixed with 35 μl of hybridization buffer ( 10 × ssc , 0 . 1 % sds , heated to 60 ° c . ), 14 μl of dd water , and 7 μl of the detection strand ( concentration of 100 pmol / μl ). this resulted in a total hybridization volume of 70 μl for each multiplexing case . from this , 20 μl of the hybridization solution for each multiplexing case was deposited over the conjugated barcode spots on the microwell chip and incubated at 40 ° c . for 20 minutes . the microwell chip was then submerged in 10 ml of washing buffer ( 0 . 5 × ssc , 0 . 1 % sds , heated to 40 ° c . ), washed by agitation for 20 s , washed again in another 10 ml of washing buffer to further reduce non - specific binding , and then let dry for 5 minutes before being imaged . note that care must be taken so that the washing buffer does not dry and crystallize over the sample spots . all images were acquired using the iphone ™ 4s from apple ( unless otherwise specified ), mounted on an olympus ix70 inverted microscope at 10 × magnification for all assays ( 10 × objective , na = 0 . 30 ) or 32 × magnification for all photographs ( 20 × objective , na = 0 . 50 , with 1 . 6 × further magnification ). quantum dot barcodes and alexa647 fluorophore were excited using a mercury lamp attached to the microscope , through excitation - emission filter sets [ λex = 350 / 50 , λem = 430lp ] ( thorlabs ), [ λex = 480 / 40 , λem = 530 / 10 ] ( thorlabs ), [ λex = 480 / 40 , λem = 580 / 10 ] ( thorlabs ), [ λex = 480 / 40 , λem = 640 / 10 ] ( thorlabs ), and [ λex = 620 / 40 , λem = 692 / 40 ] ( semrock , brightline cy5 - 4040a ). the emission filters λem = 530 / 10 , λem = 580 / 10 , and λem = 640 / 10 corresponded with quantum dots qd540 , qd589 , and qd640 , respectively , and were used to isolate for their fluorescence for resolving barcodes . the emission filter λem = 692 / 40 was used to isolate for the detection strand alexa647 fluorophore fluorescence as a means to 270 measure the amount of analyte that hybridized with its corresponding capture strand . image exposure times , made adjustable with the use of the nightcap app from apple &# 39 ; s app store , were 1 / 20 , 1 / 5 , 1 / 5 , 1 / 5 , and 1 s for the emission filters λem = 430lp , λem = 530 / 10 , λem = 580 / 10 , λem = 640 / 10 , and λem = 692 / 40 , respectively . a custom - made algorithm was written in mathwork &# 39 ; s matlab for all image analysis . the algorithm accepts as inputs five emission filter images ( λem = 430lp , λem = 530 / 10 , λem = 580 / 10 , λem = 640 / 10 , and λem = 692 / 40 ) that include samples and the same filter images of the microwell chips without beads for background intensity adjustment . the images were cropped to include beads of interest based on user selection . the cropped filter images were aligned with the λem = 430lp filter image through the use of the discrete fourier transform registration ( 21 , 22 ). the algorithm then identified the size and location of each bead , based on its appearances in the λem = 430lp filter image , using the hough transform ( 23 , 24 ). each bead was then associated with the mean pixel intensity across its area at each of the four remaining filter images . for each bead , the λem = 530 / 10 , λem = 580 / 10 , and λem = 640 / 10 filter image intensities comprised its intensity profile , while the λem = 692 / 40 filter image intensity indicated the secondary probe intensity . in order to identify the barcodes on the chip , known barcode intensity profiles were first established ( fig6 ). these profiles were obtained by imaging the barcodes b1 to b7 ( see fig8 ) alone and calculating the median filter intensity across all beads for each filter . a bead &# 39 ; s intensity profile was then compared against each known barcode &# 39 ; s intensity profile to identify the barcode of interest . specifically , a barcode was first coarsely classified according to its highest intensity among the filters λem = 530 / 10 , λem = 580 / 10 , and λem = 640 / 10 . euclidean distances between the bead intensity profile and the known barcode intensity profiles were calculated : d n = euclidean distance between the bead intensity profile and barcode n ( b1 to b7 ) intensity profile . = intensity of bead at filter m ( λem = 530 / 10 , λem = 580 / 10 , λem = 640 / 10 ). ibnfm = intensity of barcode n ( b1 to b7 ) at filter m ( λem = 530 / 10 , λem = 580 / 10 , λem = 640 / 10 ). the barcode of interest was identified as the barcode whose known intensity profile resulted in the smallest euclidean distance . finally , the median assay intensity ( i . e . λem = 692 / 40 filter intensity ) was calculated for all beads with the same barcode , and defined as that barcode &# 39 ; s hybridization signal . note that the corresponding background intensities were subtracted from 305 the recorded intensities to adjust for possible intensity variations inherent in the chips or excitation source . the secondary probe intensities were further subtracted by their corresponding barcodes &# 39 ; blank signal at the intensities from λem = 692 / 40 filter ( black bars of fig5 ). we evaluated whether the camera from a wireless communications device , in this case a smartphone , is capable of imaging the different fluorescent emitting barcodes , and whether a custom algorithm can be used to differentiate the optical signal from the secondary fluorescent label . we first confirmed that a smartphone camera had the imaging resolution and sensitivity to identify each of the barcoded beads on the chip . we designed five uniquely fluorescent quantum dot barcodes and assembled them on the surface of the chip . these barcodes contained quantum dots emitting at wavelengths of 540 , 589 , and 640 nm mixed in various ratios ( fig8 ). we placed this chip on the surface of a microscope stage , excited with hg lamp ( λex = 350 / 50 ), collected the optical signal with an objective ( 20 × at na = 0 . 50 ), filtered the emission ( λem = 430lp ), and imaged using an apple iphone ™ 4s smartphone ( exposure time = 1 / 20 s ) attached to the eyepiece of the microscope . fig1 clearly shows the ability to visually discriminate the fluorescence emission of the different barcodes . we then developed an algorithm using mathworks &# 39 ; matlab that can identify barcodes and the secondary probe &# 39 ; s signal . the algorithm accepted as inputs five emission filter images ( λem = 430lp , λem = 530 / 10 , λem = 580 / 10 , λem = 640 / 10 , and λem = 692 / 40 ) that included samples and the same filter images of the microwell chips without beads for background intensity adjustment . by way of example only , our system consists of an apple iphone ™ 4s smartphone mounted onto the front port of a microscope and a mercury lamp to excite the barcodes on the chip . the algorithm is designed to identify the barcodes by comparing the optical signal of each microbead in the wells to that of a known panel of barcodes ( see fig5 ). with reference to fig2 a , green 210 , yellow 212 and red 214 barcodes ( identified as b1 , b4 , and b6 in fig8 respectively ) were deposited on the chip and imaged using an iphone ™. fig2 a demonstrates that an iphone ™ camera is able to capture the distinct optical emission of each barcode in the well . after the assay , the smartphone camera - acquired fluorescence image of the microbeads bound with the target analyte and secondary probe ( fluorescence microscopy parameters : objective of 20 × at na = 0 . 50 , λex = 640 / 40 , and λem = 692 / 40 , exposure time = 1 s ). both green and red beads had positive signals . this demonstrates that t1 and t6 genomic targets are present in the sample but not t4 . of note , the white spots on the barcodes are due to overexposure from the high combined intensity of the alexa647 fluorophore and the 640 nm quantum dots impregnated within b6 . fig2 b shows that the barcodes &# 39 ; optical signals can be differentiated from the secondary probe using proper filtering . we next compared the analytical performance of the iphone ™ camera with an expensive charge - coupled device ( ccd ) camera in detecting target analytes on a chip . by way of example , using a model genomic sequence , we illustrated the sandwich architecture of the final microbead complex for a positive detection . we prepared a chip containing a single fluorescing barcode that was conjugated with the sequence 5 ′- gag acc atc aat gag gaa gct gca gaa tgg gat - 3 ′. we added a solution containing the target sequence 5 ′- cgg cga tga ata cct agc aca ctt a cta at ccc att ctg cag ctt cct cat tga tgg tct c - 3 ′ and an alexa647 dye labeled secondary sequence 5 ′- alexa647 - taa gtg tgc tag gta ttc atc gcc g - 3 ′. for a positive detection , the optical signal from the microbead comprises the quantum dots in the barcode and the alexa dye . the target sequence would hybridize to both the secondary probe and the barcode , and that the fluorescence intensity of the alexa dye identifies the concentration of the target analyte . we showed that an iphone ™ camera produced a similar limit of detection and dynamic range compared to the expensive ccd camera ( see fig6 ). these studies confirmed that an iphone ™ camera can image barcodes on the chip surface , be used as a detector for biological assays , and can reduce the cost and size of a quantum dot barcode detection system . the only other study using a phone camera for molecular detection of infectious diseases did not provide analytical curves to evaluate the performance of the lateral flow assay ( 5 ). lateral flow assay also cannot be multiplexed and therefore , this technique is limited to detecting a molecule that is present at thigh concentrations . while lateral flow systems are preferentially used in developing countries due to their simplicity for detection of disease markers , they typically have an inferior limit of detection in the range of mm to μm , and have limited capacity in analyzing multiple biomarkers simultaneously . a key advantage of quantum dot barcodes is that the different colors and intensity combinations of quantum dots inside the microbeads can produce a large library of barcodes , providing significant multiplexing capabilities . nie and co - workers estimated that 10 , 000 to 40 , 000 different barcodes could be generated using 5 to 6 different emitting quantum dots ( 14 ). by way of example , we selected genetic targets for influenza a viruses h1n1 , h3n2 , and h5n1 , and 20 hepatitis b and c to demonstrate the use of our integrated wireless communications device quantum dot barcode chip system for multiplex detection . the influenza a viruses are airborne , highly contagious , share similar symptoms , have posed significant difficulty in clinical differential diagnosis , and remain pandemic risks ( 8 , 9 ). the blood - borne viruses hepatitis b ( hbv ) and hepatitis c ( hcv ) are prevalent in resource - limited settings ( 10 ). these infections are difficult to differentiate clinically since they share common symptoms such as general malaise , jaundice , and nausea and / or vomiting ( 11 ). we designed seven barcodes for each of five infectious disease biomarker targets plus a negative and positive control ( see fig8 ). the analytical sensitivity and linear dynamic range for each of the barcode ( see fig2 c ) is on average 50 fmol and up to 100 - fold , respectively . here we demonstrated that our chip is able to detect multiple biomarker targets simultaneously . we prepared six different mock genetic samples by mixing different combinations of the genetic target sequences for each of the five pathogens of interest plus a positive control sequence , and a secondary fluorescent probe sequence . for example , we would prepare solutions that were spiked with the target sequences t1 , t3 , t5 , and t7 , or t2 , t4 , and t7 . a sample of 10 μl was added to the chip and incubated at 40 ° c . for 20 minutes , rinsed with a washing buffer , dried , imaged , and analyzed using the algorithm . fig3 shows that we can identify all the target biomarkers in solution for all six samples . for example , in fig3 b , our solution contained the sequences t1 , t3 , t5 , and t7 and the bar graph shows our technique can discriminate between barcode containing a secondary probe versus those that do not ( t2 , t4 , and t6 ). it has been demonstrated herein that the combination of quantum dot barcodes with wireless communications device technologies to engineer a device capable of detecting different types of targets , in this example , infectious diseases markers . there are two major inventive aspects to the present invention : ( a ) barcodes can now be easily transported on a chip and ( b ) integration of barcodes with portable wireless communications device technology enables multiplex detection anywhere in the world without the need for skilled technicians to interpret the data . this detection device enables hospitals , environmental control agencies , disease control centers , and the military to monitor the onset and spread of contaminants , pathogens and other targets of interest and it can be used at point - 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