Patent Application: US-201214001927-A

Abstract:
the present invention provides a sensor array device with multiple sensor junctions which have been created through the assembly of two or more differently functionalized surfaces . the functionalizing of the prospective sensor junction areas with sensor compounds occurred when the different surfaces were physically separated from each other before the assembly of the sensor array . by these means , sensor junctions can be built smaller than conventional deposition techniques like printing and photolithography would allow for otherwise . as a consequence , each individual sensor junction contains two potentially different sensor compounds . the sensor array identifies and quantifies different biomolecules .

Description:
preferred embodiments of the present invention will be described in detail below with reference to the drawings . first preferred embodiment : sensor arrays with conducting sensor half - elements which are supported by plates and contain dna sensor compounds the first preferred embodiment is shown in fig1 and 2 and describes a sensor array 12 with linear conducting sensor half elements 1 , 2 , which are supported by two common carrier 61 , 62 formed by plates . in this preferred embodiment of the invention , the sensor compounds are comprised of or comprise oligonucleotides . the analyte entering the measurement cell ( not depicted ), which surrounds the sensor array 12 , is a mixture of cdna molecules . the junction areas 31 ( cf . fig3 ) are part of elongated spots , which are arranged along rows and columns of sensor half elements 1 , 2 . all sensor half elements 1 , 2 are arranged in parallel on their respective common carriers 61 , 62 . one common carrier 61 , 62 is depicted in fig1 . the functionalization of the sensor half elements 1 , 2 occurred before assembling the common carriers 61 , 62 . during assembly the first common carrier 61 and the second common carrier 62 are approached to each other in such manner that the row and column elements 1 , 2 contact each other and are oriented in an angle of 90 degrees as shown in fig4 . the common carriers 61 , 62 are made from metal coated silicon wafers using a standard photolithographic process . the preferred design consists of 1000 parallel conductors 74 of 2 . 5 cm length and 20 μm width which are separated by 5 μm . the individual conductors 74 serve as carriers 7 for the sensor half elements 1 , 2 . the conductors 74 are arranged on a square shaped area on the respective common carriers 61 , 62 . the common carriers 61 , 62 have an edge length of 2 . 5 cm . the conductors 74 feature a semi - circular cross - section and have the form of a half cylinder , fig1 . the length , width and spacing of the conductors 74 can be easily adapted to the number of sensor half elements 1 , 2 and the number of desired sensors 3 . the shape of the sensor half elements 1 , 2 which are created on the common carriers 61 , 62 may alter . an alternative preferred embodiment comprises trapezoidal conductors 74 with crenated surfaces . in both cases sensor half elements 1 , 2 have at least one microscopic convexly shaped surface area at and around the point of contact another sensor half element 1 , 2 . accordingly , parts of the surface can be porous or wavelike structured . the surfaces of the conductors 74 are coated with an insulating thin layer 82 or film by sputtering as part of the photolithographic process that is used for the formation of the common carriers . the sensor half elements 1 , 2 are arranged in such manner that the surfaces of the row elements 1 are touching the surfaces of the column elements 2 as shown in fig2 . the insulating layer 82 prohibits a direct electrical contact with another conductor 74 . the thickness of the non - conducting insulating layer 82 is typically within the submicron range . the insulation layer 82 forms a permeation barrier for electrons and ions . the thickness of the insulating layers 82 of two sensor half elements also determines the closest distance between the conductors 74 and the dimension of the gaps . in this embodiment , the insulating layer 82 is made of polyurethane . alternatively , it is also possible to use nitrides , oxides and other chalcogenides , self - assembled monolayers , polyelectrolyte multilayers , polymers like polyimides or fluoropolymer - copolymers , electro dipping varnishes , or others known to the art instead . glass can also be used as insulating coating . another alternative preferred embodiment utilizes common carriers 61 , 62 with spacers 65 where the spacer elements 65 have been integrated into the common carrier plates 61 , 62 as it is shown in fig3 . those common carriers 61 , 62 are flat silicon wafer substrates with a pattern of 5 . 1 μm high pillars as spacers 65 . a standard cmos processes crafts all conductors 74 by physical vapor deposition as 2 . 5 μm high , 20 μm wide and 10 mm long lines . the center to center distance of the lines is 25 μm . 250 lines are set in parallel covering a total width of 6 . 25 mm . first , 5 nm chromium undercoating is deposited onto a developed photoresist mask before depositing 2 . 495 μm gold conductors . after removing the mask a reactive plasma coating process deposits a flat insulating layer of sio 2 82 above the common carrier 6 including the sensor half elements 1 , 2 and spacers 65 . here , the surface of the sensor half elements 1 , 2 does not necessarily have to be convexly shaped and can be entirely flat as shown in fig3 because the spacers 65 have a defined thickness and prevent the sensor half elements 1 , 2 from touching after completing the sensor array assembly as shown in fig4 . the spacers 65 fulfill two functions , namely to firstly create a tiny gap which allows the analyte to enter the gap region and secondly to prevent the conductors 74 from touching each other to avoid short circuits . even though the passivation and / or insulation of the conductors 74 is possible , those sensor half elements 1 , 2 do not necessarily require an insulation layer 82 . the photolithographic process is also used to integrate the addressing or multiplexing units 111 , 112 directly into the common carriers . the layout ensures that the conductors 74 are connected to the selection units 111 , 112 as schematically shown later in fig9 . either the carrier 7 itself , the insulating layer 82 , or an additional carrier material layer 8 may contain the sensor compounds 4 , 5 . in the preferred embodiment of the invention , a carrier material layer 8 is applied upon the insulating layer 82 . the carrier material layer 8 is able to covalently bind the sensor compounds 4 , 5 . in the following example , the carrier material layer 8 contains side chains with functional groups which are compatible for cross - linking to appropriately modified oligonucleotides either directly or mediated through an activation reagent . 1 - ethyl - 3 - 3 - dimethylaminopropyl carbodiimide , edac is able to cross - link carboxy groups with amines , glutaraldehyde , bissuccinimidyl esters , diisocyanates or diacyl chlorides cross - link amines with amines , or the formation of thioether cross - links through thiol - reactive groups at amine sites by succinimidyl trans - 4 - maleimidylmethylcyclohexane - 1 - carboxylate . preferred substances for sensor compounds 4 , 5 are oligonucleotides with sequences which are suitable to function as hybridization probes or in particular as primers for solid phase pcr . one preferred class of such sequences has been described in wo2007062445 [ seitz , 2007 ]. herein , the oligonucleotides are able to specifically react , which means hybridize and prime , with start and end sites of polynucleic acid analytes 9 . other preferred classes of sequences are gene and transcript specific primers . for example such sensor array is designed to target blastoma associated gene expression . for this purpose , 84 glioblastoma associated genes with regulated alternative splicing candidates and putative chimeric transcripts , 9 astrocytoma associated intergenic transcriptionally active regions , 33 control regions with housekeeping genes , transcriptionally silent genomic areas , brain and liver associated candidate genes are selected . genespecific primer pairs are designed for all regions of interest which include several primer pairs per gene , one for each exon and one for each exon - exon junction , which lead to a total of 1794 individual primers . redundancy which means that certain primers can be used in different primer combinations results in 15507 unique primer combinations . furthermore , single and , no primer amplification as well as background controls based on non - related genomic regions from mouse and e . coli complement the set of sensor compounds . it leads to a total of 15625 primer combinations . quadruplet repeats bring the total of sensor junctions 31 to 62500 which corresponds to 250 row and 250 column sensor half elements . it represents 1 / 16 th of one larger sensor array with 1000 × 1000 sensor half elements . all primers are designed to comply one melting temperature tm of 62 ± 0 . 5 ° c . the primers contain 21 to 29 nucleotides and have no homopolymeric stretches exceeding three consecutive nucleotides . the primers contain a 5 ′- amino group modification to be able to covalently bind to the solid support . the sensor compounds 4 , 5 are immobilized to the surface of the carrier material layer 8 by covalent binding to the surface via cross - linkers which were described above . one separate sensor half element 1 , 2 is depicted in fig5 . it consists of a carrier 7 , which is covered by an insulation layer 82 and material layer spots 8 which contain the sensor compounds 4 , 5 . the sensor half elements 1 , 2 are either functionalized separately or alternatively , the sensor half elements 1 , 2 are assembled on common carriers 61 , 62 and functionalized afterwards in groups which are determined through the assembly on the common carriers . however , before the common carriers 61 , 62 are approached the sensor half elements 1 , 2 are functionalized on the respective common carriers 61 , 62 . if the carriers 7 of the sensor half elements 1 , 2 are narrowly spaced on the respective common carriers 61 , 62 before functionalization , the functionalization feature a precise lateral resolution of the same dimensions as the sensor half element assembly . in order to obtain such lateral resolution , three technologies fulfill those requirements . firstly , standard piezo plotters with spot sizes at around 20 μm are able to deposit the sensor compounds 4 , 5 from diluted solutions , e . g . 20 μm , onto the respective positions of the carriers 7 which are covered by the insulating layer 82 and material carrier layer 8 . printing is followed by incubation at constant humidity and elevated temperatures , e . g . 60 ° c ., which facilitates the covalent binding to most of the available binding sites . afterwards , any surplus sensor compounds 4 , 5 which have not reacted are removed by flushing with blocking and washing solutions . as a result , the localized areas of the sensor half elements 1 , 2 are covered with dense material carrier layers 8 containing the dedicated sensor compounds . secondly , for line widths below 50 μm dna probes can be directly synthesized or on the surfaces of the respective carriers 7 . this process is directed by photolithography and uses photo - activatable linkers . it is already a standard technology in microarray production . only the sensor substances 4 , 5 , e . g . partial sequences of the dna probes which are specific to the individual sensor half elements 1 , 2 have to be synthesized at the surface . common sequence motifs of the sensor compounds 4 , 5 can be synthesized beforehand in bulk syntheses and immobilized to the insulating layer 82 or carrier material layer 8 . such pre - cursor compounds can be applied unison to many or all carriers 7 at once . alternatively , it is also possible to modify all or groups of sensor half elements 1 , 2 with precursors before the remaining nucleotides are synthesized step by step in situ . the individual nucleotides can be deposited using microcontact stamps . because the genetic code contains four different bases only four different stamps are required for one additional nucleotide position . each reaction is followed by a washing and activation cycle . it is also possible to immobilize a series of precursors , e . g . 64 which contain three selective nucleotides already . afterwards , only a reduced number of additional selective nucleotides is synthesized in situ at the surface . thirdly , stamps which contain microfluidic channels are filled with the respective compounds 4 , 5 . for instance , the stamps contain 64 channels . 16 such stamps comprising 64 channels respectively are used in line to modify up to 1024 sensor half elements 1 , 2 . this method is advantageous if entire sensor half elements 1 , 2 are modified with the same sensor compound 4 , 5 . for example , the entire surface of the common carrier plates 61 , 62 including the rows and columns of conductors 74 , which have been protected by the insulating layer 82 sio 2 , are activated by immersing the surfaces in silanization solution of 3 % 3 -( glycidyloxypropyl ) trimethoxysilane in 95 % ethanol . the silanization produces a homogeneous reactive layer which is stabilized through moderate baking at 110 ° c . such activated surfaces can be stored under inert gas or in vacuum . a number of 897 primers of the above example are spotted using a piezo printer to each common carrier 6 along the lines of sensor half elements 1 , 2 in a pattern of randomly distributed triplicates . the spotting layout for both common carrier plates 6 match the desired primer pairs in the finished assembly . forward and reverse primers are distributed to the sensor half - elements 1 , 2 on both common carrier plates 61 , 62 . the diameter of a single spot is 20 μm , the pitch is 25 μm , and 250 × 250 regular spots are set into a square format . the covalent binding proceeds in a humidity chamber over night at 25 ° c . not reacted molecules and spotting buffer are removed through immersion in blocking solution of 50 mm ethanolamine and / or 100 mm tris at ph 9 for 15 minutes followed by thoroughly rinsing the surfaces with water . alternatively , it is possible that another insulating layer 82 is directly functionalized with the sensor compounds 4 , 5 . such an embodiment of the invention does not contain an additional carrier material layer 8 . the insulation layer 82 itself binds or contains the sensor compounds 4 , 5 . further alternatives are that the sensor compounds 4 , 5 can be embedded or bound to the carrier material layer 8 during the coating process , e . g . when the insulating layer 82 is coated with the carrier material layer 8 . however , sufficient amounts of sensor compounds 4 , 5 remain accessible at the surface . in the preferred embodiment of the invention all sensor half elements 1 , 2 are made from the same basis material and each of the used carriers 7 or conductors 74 can be individually modified using the same chemistry . the sensor half elements 1 , 2 are processed separately in order to ensure that the sensor compounds 4 , 5 remain physically separated during their immobilization . alternatively , it is also possible to use different basis materials and / or different binding chemistry . for instance , different organic molecules like amino - or carboxy - compounds can insert different lateral spacers at the surface of row elements 1 and column elements 2 to design junction areas 31 with defined surface charge asymmetries . assembly of the two sensor half - elements to one sensor array and measurement cell each of the sensor half elements 1 , 2 on the common carriers 61 are prepared in the above mentioned manner . afterwards , the common carriers 61 , 62 are assembled to the sensor array 12 which has the form of a cross network as shown in fig2 or 4 . both common carrier plates 61 , 62 with the sensor half elements 1 , 2 are stacked in such way that the sensor half elements 1 , 2 located on opposing common carriers 61 , 62 face each other , whereby each two sensor half elements 1 , 2 facing each other are oriented perpendicularly . the spacers 65 in fig4 ensure a defined gap of 100 nm between the two sensor half elements 1 , 2 for each junction 31 . each junction 31 constitutes one individual sensor 3 . schematic close - ups of the formed junctions are shown in fig6 and 7 . the figures do not represent the correct spatial relationships between the conductors 74 , which have cross - sections of one to several μm , whereas the coatings are in the submicrometer range , the exemplary molecular sensor compounds 4 , 5 and analytes 9 such as dna molecules . the latter typically have an average length of 2500 nucleotides and are approximately 0 . 85 μm long . the assembly becomes embedded into a cartridge enclosure which provides microfluidic connectors , sealing along the edges , electric and thermal contacts to the carrier plates . the basic principle of the sensor action is a molecular recognition reaction between the analytes 9 under investigation and the sensor compounds 4 , 5 . different kinds of reactions are possible , for example : fig6 to 8 show schematic representations of the reactions a and b . the symbols indicate a certain nucleotide adenine , a , thymine , t , guanine , g or cytosine , c which are able to match the complementary nucleotide , a with t and g with c . the filled symbols represent the analytes whereas the hollow symbols stand for the sensor compounds or reaction products . the arrows indicate the 5 ′ towards 3 ′ direction of the oligo - and polynucleotides . sensor 4 , 5 compounds are oligonucleotides which are immobilized either with their 5 ′- site at one group of sensor half elements , e . g . at all row elements 1 shown as the top element in fig6 , or with their 3 ′- site at the opposite column elements 2 shown as the bottom element in the same figures . molecules of an analyte mixture 9 of e . g . cdna which enters the sensor array are able to hybridize to the sensor compounds 4 , 5 as shown in fig6 . analytes hybridize at the 3 ′-, 5 ′-, both or non sites . the hybridization buffer , containing e . g . de - ionized formamide , denhardts , tween , sds , dextran and depc , the temperature and time are optimally chosen for sequence specific dimerization and trimerization . such conditions are based on the primer design e . g . 62 ° c . and 1 h . during this period the solution is agitated to accelerate diffusion rates into the surface of the gap regions . the final hybridization is the equilibrium binding state . not hybridized polynucleotides can be removed through washing steps . the result is a segregated analyte pattern in the sensor array . the sensor compounds might contain a position lock , which means that sequences extensions of analyte and the sensor compounds allow hybridizations only at the start 911 or end 921 of the analytes . the consequence is that short sequences of 1 to 8 nucleotides can be used to obtain an efficient segregation . for illustration , each longer analyte nucleotide sequence has anywhere in their chain at least one a , but only in average one quarter starts with one a . this principle applies to each nucleotide position . sensor compounds 4 , 5 are primer oligonucleotides , which are immobilized with their 5 ′- site as shown by sketches in fig8 . in a first step i ) in fig8 , polynucleotides of the analyte mixture 9 entering the sensor array 12 and hybridize only to those primers 4 , 5 which are complementary to their 3 ′- side . the hybridization buffer contains deionized formamide , denhardts , tween , sds , dextran and depc , the temperature and time are optimally chosen for sequence specific hybridization , based on the primer design for example 62 ° c . for 1 h . during this interval the solution is agitated to accelerate diffusion rates into gap regions . non - hybridized analytes 9 can be removed by washing . in a second step ii ), an assay with activated polymerase like taq , pfu , phusion or similars , single nucleotides dntp &# 39 ; s and additives such as divalent cations and stabilizers are applied to perform a single elongation 70 ° c . for 2 min . the reaction results in bound complementary copies of the analytes 9 at the sensors 3 according to the sequence and proportional to the starting concentration . stringent washing denatures the dsdna and the original template can be removed leaving the covalently bound complementary sequence at the sensor array 12 . in a third step iii ), a fresh assay with polymerase , dntp &# 39 ; s and additives will be inserted and a controlled polymerase chain reaction is performed through thermocyling , e . g . 50 cycles of 95 ° c . for 30 sec , 62 ° c . for 30 sec and 70 ° c . for 2 min . only those polynucleotides which find complementary primers at the opposite surface can be amplified . the amplifications proceed alternately between the opposite surfaces in the gap regions . the hybridization method , a , is simple because no additional enzymatic reaction and no fast thermocycling is required . the hybridization and amplification method , b , is technically more sophisticated but provides two advantages . first , the proof - reading function of the polymerases introduces corrections for mispriming events . second , the amplification multiplies the amount of analyte 9 in the junctions 31 through the generation of identical copies . the biochemical molecular recognition fulfilled the first part of the sensor reaction . it detects and segregates the analyte 9 into different subpools . it means that each sensor 3 contains predominantly sequences which correspond to the sensor substances 4 , 5 of both sensor half elements 1 , 2 facing each other . a real time or endpoint measurement can only identify the amount but not the kind of material in each sensor 3 . with regard to subsequent electrical characterizations further signal enhancements can be achieved through post labeling with materials which possess strong interaction with alternating electric fields for example conjugated polymers or metallic nanoparticles . the primary sample can be rna extracted from a tissue which has been obtained by biopsy . at the time of sampling , the tissue has been immersed immediately in rnalater which is an aqueous storage reagent that rapidly permeates tissues to stabilize and protect cellular rna . a kit is used to generate cdna as secondary sample through reverse transcription , rna digestion and purification . in this process is the sample can be modified through sequence extensions which enable primers to lock into respective start and end site positions . the processing follows the above described principle . for the hybridization and amplification assay a minimum of 1 μl of cdna sample and pcr master mix which includes dntp mix , polymerase , buffer and additives , is injected via the microfluidic ports into the gap between the sensor half - elements 1 , 2 . heating for 30 sec to 94 ° c . melts and unfolds any hybridized polynucleotide stretches and activates the polymerase . the initial hybridization phase , i ) in fig8 , just above the annealing temperature of the given example , 62 ° c ., enables the cdna templates , shown with black filled symbols in fig8 , to bind to the corresponding sites at the sensor compounds , shown with hollow symbols in fig8 . one elongation step for 1 minute at 74 ° c . generates surface bound complementary copies of the analytes , ii ) in fig8 . the following denaturing step , 30 sec at 94 ° c ., and washing step removes any analyte molecules , which is symbolized in step ii of fig8 through the dashed line of the cdna template and the arrow which indicates that those molecules leave the system . during the subsequent annealing phase , 30 sec at 62 ° c ., hybridize surface bound analyte copies with sensor compounds at the opposite sensor half element surface , iii ) in fig8 . only those molecules are able to amplify during the next pcr cycles . a minimum of 1 μl fresh pcr master mix is injected . pcr is performed over 50 cycles . the dna concentration increases with each cycle in those junctions where analyte molecules with end sequences compatible to both sensor compounds 1 , 2 have bound during the first hybridization phase . as the carrier of the sensor half elements 1 , 2 is conductive each junction 31 can be electrically addressed through both of the connecting sensor half elements 1 , 2 . the measurement of amplified dna at each junction 31 is performed by impedance measurements either after each cycle at constant temperature or as endpoint measurement . the junction areas 31 act as capacitors which are able to sense the dielectric properties of the compounds in the gap region or the sensor 3 . each junction area 31 can be envisaged in a first approximation as a parallel alignment of tiny plate capacitors of different width and area which enclose small partial volumes adding all up to the total active surface and volume . the detection of the capacitance occurs through sending a timely variable electrical signal , e . g . voltage steps and pulses , ac potential or current , along one conductor and recording the response at the other conductor . the relative permittivity of dna solutions changes in a concentration dependent manner from pure water with a relative permittivity , ∈ r of 80 to over 90 for a 1 % dna solution . those values have been measured for example at 1 mhz by takashima [ 1984 ]. in contrast , air has a ∈ r - value of 1 whereas typical organic polymers range between 6 and 10 . this implies that double - stranded dna strongly alters the dielectric response of the sensor junctions . impedance changes are recorded during the measurements . the results of the measurement are quantified during the measurement series and the data analysis recalculates the concentration ratios of the analyte molecules 9 in the original sample . in first approximation , each sensor 3 can be envisaged as a parallel alignment of tiny plate capacitors of different width and area , which all add up to the total active surface area and volume . for example , a 50 μm conductor 74 junction 31 forms a total cross - section of 2 . 5 · 10 − 9 m 2 . with an insulating layer 82 of 0 . 1 μm thickness , the minimal separation of both sensor half elements 1 , 2 is 0 . 2 μm . using the following equation with 10 equal steps and ∈ r of water with 80 , the total capacitance approximates to 192 . 1 ff . assumed that the most inner part of the junction 31 is defined through the first 10 th of the distance and area changes its dielectric properties due to an accumulation of dna following above described molecular recognition reactions . if ∈ r of this section changes from 80 to 90 , the capacitance increases to 200 . 3 ff . fig9 shows the evaluation circuit 100 comprising a first selection unit 111 , e . g . an analog multiplexer , with one primary port and a plurality of secondary ports 114 which are individually connected to one row element 1 each , a second selection unit 112 with one primary port and a plurality of secondary ports 115 which are individually connected to one column element 2 each . the circuit 100 further comprises a control circuit 120 which controls the two selection units 111 , 112 . the first selection unit 111 selects one of the row elements 1 and the second selection unit 112 selects one of the column elements 2 . the measurement circuit 130 quantifies the electrical impedance between the main ports of the addressing circuits 111 , 112 . each combination of a column element 2 and a row element 1 enables one distinct sensor 3 formed between the respective selected sensor half elements 1 , 2 . the impedance of the addressed sensor 3 is measured by measuring the impedance between the primary ports of the selection units 111 , 112 . sensor half elements 1 , 2 next to the addressed combination of sensor half elements 1 , 2 are held or fixed to a constant potential , e . g . floating ground . all sensors 3 are measured in consecutive or any other order . it is possible to analyze several sensors 3 in parallel by addressing one row sensor half element 1 and several column sensor half elements 2 , and vice versa , by employing a number of further selection units 111 , 112 and measurement circuits 130 . in this preferred embodiment of the invention , the measurement unit 130 is a combination of potentiostat and frequency analyzer that records amplitude and phase shift of the response signal in comparison to the entrance ac signal . impedance spectra can be used to characterize the substances being bound to the respective sensors 3 . oscillator , charge and ac bridge based approaches are in use to determine capacitances , whereby the charge based capacitance measurement technique , cbcm , is the simplest realization . ac bridge based commercial instruments and circuit designs are available to measures capacitances with 1 ff resolution but potentiostats have been presented to approach the 10 af range [ carminati , 2009 ]. it might be not always necessary to determine capacitances or impedances through modulus and phase shift . under equilibrium conditions both readings can be transformed into each other . furthermore , if a phase shift is seen to be known it might be enough to determine the modulus and calculate the capacitance using a valid equivalent circuit . by these means is it possible to use single frequency measurements to determine valid capacitances . the impedance or capacitance is either determined by end point measurements or by using real time measurement . real time measurements are able to characterize the temporal behavior of the electrical properties of the sensors during the molecular recognition reactions . real time therefore enables to follow hybridization kinetics and amplification rates during thermocyling depending on the type of experiment . for endpoint measurements is it advantageous to wash the sensor array with pure water and also measure in pure water or under dry conditions . it reduces the cross - talk between neighbouring sensors 3 . second preferred embodiment : sensor arrays with transparent sensor half element glass fibers and polypeptide sensor compounds the second embodiment of the invention describes a sensor array 12 with straight aligned sensor half elements 1 , 2 as shown in fig1 or woven sensor half elements 1 , 2 as shown in fig1 which are grouped as row 1 and column elements 2 . all sensor half elements 1 , 2 are identical except their individual functionalizations . the gap between the sensor half elements 1 , 2 is caused by the convexly shaped surface of the sensor half elements 1 , 2 . the sensor half elements 1 , 2 are transparent . an optical readout device 141 detects changes in the gap area 31 of the individual sensors 3 . the sensor compounds 4 , 5 are polypeptides and polynucleotides . the analyte 9 are polypeptides . commercially available glass fibers of 20 μm outer diameter were chemically activated by silanization to produce amino - reactive layers with n - hydroxysuccinimide , nhs activated moieties in a batch process . twenty antibodies like anti - tbp mab and anti - crebbp mab which are directed against known human transcription factors were covalently bound to the nhs surfaces of the glass fiber carriers . for this purpose , a relief printing plate has been made from standard photopolymer printing resist with 20 μm broad elevated lines which were charged with solutions of said antibodies in immobilization buffer at approximately 1 μg / μl . the antibodies serve as sensor compound 4 and are shown as y - shaped symbols in fig1 . the deposition of antibodies in the designated areas along a first batch of fibers has been achieved by contacting activated fibers and the printing plate in perpendicular orientation relative to the elevated stamp profile . the fibers were incubated until the binding reaction has completed . subsequent hydrolysis of non reacted nhs - ester affects the unmodified stretches of the glass fibers to be hydrophilic . as consequence , functionalized fiber areas are interspersed by hydrophilic but unmodified regions . fibers are at least 1 cm long and can be stored until device assembly . a second batch of fibers is modified using a selection of presumed dna sequences responsible for transcription initiation . the sequences include regions upstream of transcription start sites and a 5 ′- amino modification . the sequences serve as second sensor compounds 5 , were also immobilized by covalent to nhs - activated glass fibers as described above and are shown as double line in fig1 . assembly of the sensor half elements to one sensor array and measurement cell the sensor array 12 comprises the entirety of all individual junction areas 31 . for practical reasons such as adding the mixture of target compounds or flushing the sensor array with washing buffer such sensor array 12 is supported and / or encapsulated by a frame , chamber , cartridge or other kind of enclosure . such assembly constitutes the measurement cell . measurement cells are preferably manufactured from polycarbonate . the sensor half elements 1 , 2 are modified fibers featuring a length of some centimeters that are mounted to the cell void at designated places . a robotic mini - loom assembles the row sensor half elements 1 from the first batch carrying antibodies and the column sensor half elements 2 from the second batch with dna into a woven mesh like structure as shown in fig1 . the correct positional alignment of the sensor half elements 1 , 2 or modified fibers is facilitated by tracking co - deposited dye in the functionalized fiber segments or segments of the sensor half elements 1 , 2 during assembly . the polycarbonate cell is transparent , encapsulated and contains mircofluidic ports . filling of the fiber sensor array occurs via feeder and drain channels , connectors and vents . fluorescence signals are read by a coupled fluorescence microscope . absorbance measurements are carried out by coupling the optical paths to via fibers to a spectrophotometer . the glass fibers forming the carrier of the sensor half elements 1 , 2 focus the optical path at individual junctions 31 which leads to an increased signal - to - noise ratio . at the beginning of each experiment a baseline scan of the sensor array 12 is recorded before the cell is filled with a solution containing a mixture of transcription factors , the analyte 9 , white square shaped symbols in fig1 . the transcription factors are captured by the immobilized antibodies at the row sensor half elements 1 , i ) in fig1 , and interact with column bound dna fragments to form dna - protein complexes . after a low stringent washing step another optical scan quantifies the amount of protein which has been captured by the individual sensors in the sensor array . then , a second analyte 9 sample solution with enhancer proteins is injected and coordinates with the formed dna - protein complexes , ii ) in fig1 , black disks . finally , an in - vitro transcription is carried out using rna - sensitive fluorescent dyes or fluorescent labeled nucleotides , iii ) in fig1 . the continuous observation of signal derives kinetic information on transcription factor , enhancer and dna - sequence interdependence and results in transcription initiation efficiency . alternatively , whole cell extracts can be used instead of purified enhancer proteins . the efficiency changes are quantified to conclude the transcriptional state of the analyzed cells . the preferred embodiments above describe sensor half elements 1 , 2 which hold sensor compounds 4 , 5 at localized junction areas 31 . those sensor compounds can differ of course . however , it is also possible that each sensor half element 1 , 2 contains only very few or even just one sensor compound 4 , for example one forward primer 4 or one reverse primer 5 . this embodiment implies the following two features . firstly , the number of different sensors compounds 4 , 5 on the sensor half elements 1 , 2 increases linearly while the number of different unique sensors 3 increases quadratically . in our example , each combination of one forward primer 4 with one reverse primer 5 leads to one unique sensor , and therefore the combination of m forward primers 4 and n reverse primers 5 results in m × n unique primer combinations and sensors 3 . this combinatorial sensor design principle minimizes the need for extensive primer libraries . secondly , each sensor half element 1 , 2 only needs to be coated with one sensor compound , which lowers the requirements for the deposition methods of spotting or micro contact stamping . position accuracy is less important along each sensor half element , because large parts of the whole surface of the sensor half element 1 , 2 is covered with the respective sensor compound 4 , 5 . the coating of fibers or wires can also occur entirely without spatially resolved deposition methods . such sensor half elements 1 , 2 can be functionalized in a batch process before assembling the sensor half element 1 , 2 to the respective common carrier 61 , 62 . the first preferred embodiment describes conductive sensor half elements 1 , 2 and in particular the electrical measurement of the individual sensor 3 . the second preferred embodiment describes the functionalization and the assembly of fiber sensor half elements 1 , 2 which are not supported through a common carrier 6 . according to the first and second preferred embodiment of the invention , the carriers 7 are conducting wires 74 made of metals like copper , gold or suitable alloys having the same or higher electrical conductivity . as for a high degree of integration thin and long conductors are required , high specific conductivities are advantageous to obtain an acceptable conductivity of the sensor half elements 1 , 2 . sensor half elements 4 , 5 have an insulating coating 82 . according to a first alternative of the invention , the carriers 7 are made from aluminum wires with thicknesses on the order of 10 to 50 μm being anodized to form an insulating layer 82 . current density , time and the anodizing solution determine the density and thickness of the oxide layer . then , through the reaction of organosilanes such as 3 - aminopropyltrimethoxysilane or n - 2 - aminoethyl - 3 - aminopropyl - trimethoxy - silane with hydroxyl groups , which have formed from the most outer oxide layer in aqueous phase , amino functionalization is introduced . this allows for the binding of appropriate oligonucleotides or - peptides trough cross - linking reactions as described above . according to a second alternative of the invention , the carriers 7 can be made from carbon fibers being chemically oxidized at the surface to gain a high density of carboxyl groups . those carboxyl groups are able to react with transient activation reagent like edac . amine modified oligonucleotides can be cross - linked afterwards to form an impermeable interface between the carbon fiber and the oligonucleotides or - peptides . the extent of surface interaction can be enhanced through additional soft matter coatings which are grouped in the category carrier material layer 8 . polymers , in particular gels , are suitable to form a coating which can be squeezed . such coatings contain binding sites to covalently bind the sensor compounds 4 , 5 . the junction area 31 , i . e . the region between two sensor half elements 1 , 2 where molecules 9 under investigation can bind to each of the sensor compounds 4 , 5 with two of its binding sites 91 , 92 , can be increased by using said gels . dendrimers like polypropylenimine polyamine range from tetramines to tetrahexacontamines and can be chosen to build 3d - like structures with higher interface densities of the sensor compounds 4 , 5 . fig5 presents a sensor half element 1 , 2 which is supported by a carrier 7 and coated by an insulating layer 82 . the different sensor compounds 4 , 5 are embedded in a carrier material layer 8 and are applied in separated regions or areas 76 , 77 . the position of the junction areas 31 is defined by the position of the separated areas 76 , 77 of the sensor half elements 1 , 2 . the areas should be kept as small as possible without compromising the size of the sensor area which can be connected through the analyte molecules 9 . the minimized design combines two advantages . first , less sensor compounds 4 , 5 are required to produce the individual senor half elements 1 , 2 . second , the surface regions outside the junction areas 31 , where the analyte could be trapped without contributing to the measurement , are minimized . as a consequence , the active surface is present in the junction area 31 only . one alternative to the spatial resolved modification of sensor half elements 1 , 2 is to coat and immobilize large areas of the sensor half elements 1 , 2 . afterwards the layers at regions outside the junction areas 31 are stripped . light can be used to trigger a release reaction outside the shielded junction areas 31 when using a photolabile linker to immobilize the sensor compounds 4 , 5 . in addition to standard weaves , which are made of regular warp and weft patterns , structures can be formed in which pairs of sensor half elements 1 , 2 cross each other several times . alternatively , it is also possible that several sensor half elements carry the same sensor compounds 4 , 5 . both methods lead to a built - in redundancy implying that multiple individual sensors are chemically and functional identical . by these means , the signal to noise ratio can be increased at the cost of the total integration density . such a trade - off may become important for the measurements of rare analytes with small detectable total numbers which noticeably underlie a poisson distribution . the built in parallel measurements and redundancy will increase the confidence . of course , those ratios are considered for the whole measurement process from the sample preparation up to its measurement , but also in the design of the sensor array . dielectric macromolecules like dna can be directed into the gap region 31 through non - uniform electric fields , a process which is called dielectrophoresis and which increases the local concentrations of the analyte 9 in the sensor array junctions 31 and accelerates the molecular recognition events . signal enhancements can be enhanced through post labeling with materials which show strong interaction with alternating electric fields like conjugated polymers , metallic nanoparticles or other dielectrics . experimental confirmation part i : proof - of - principle of sensor arrays with sensor half elements this experiment concerns the result shown in fig1 . the employed sensor array 12 contains sensor half elements 1 , 2 which are supported by two common carrier plates 61 , 62 . the junction areas 31 are defined as round spots which are placed on the first sensor half element 1 . the second sensor half element 2 has not been structured and consists of a homogeneously functionalized surface . the focus of this demonstration is the specific analyte 9 amplification of a 400 nucleotide long template 9 between two surfaces . the experiment demonstrates the principle of the dual solid phase amplification reaction . one n - hydroxysuccinimide activated 75 × 25 & gt ; 1 mm glass slide from a commercial supplier , polyan , germany , is the first carrier 71 and was used to immobilize amino - modified dna oligonucleotides by contact spotting . the spotting solutions contained 20 μm of individual primers or 10 μm plus 10 μm of dual primer mixtures . forward primer , reverse primer , the combination of both and a fluorescently modified guide dot oligonucleotides have been spotted to designated areas . the forward primer 4 is complementary to the 5 ′- site of the template 9 and the reverse primer 5 identical to the 3 ′- site of the template 9 . the spotting produced a number of identical arrays with spot sizes of 120 μm diameter . the spotted dna reacted for 12 hours at room temperature in a chamber of approximately 30 % humidity which has been adjusted by saturated nacl solution . then , the surface has been immersed in blocking solution of 50 mm ethanolamine and 100 mm tris at ph 9 for 15 minutes before rinsing it with water . a fluorescence scan , shown in fig1 , proved the quality of the array based on the amount of bound guide dot . the glass slide is the first sensor half element 1 , one surface which carries a separate spots of forward and reverse primer sensor compounds 4 . the mixed primers 4 , 5 and guide dot oligonucleotides are for control purpose only . for the second carrier 72 22 × 22 × 0 . 3 mm glass cover slides are used and were immersed in freshly prepared 3 % 3 - methacryloxypropyltrimethoxysilane in 95 % ethanol silanization solution . this process crafted a homogeneous reactive layer at the surface . after rinsing twice in ethanol the cover slide was baked for 15 minutes at 80 ° c . a mask has been made from a 94 μm thick adhesive plastic film consisting of a 3 . 5 mm frame and a square - shaped central void . aminoreactive polymer has been produced by liquid - phase co - polymerization of mixed polyacrylamide , bis - acrylamide and glycidyl - methacrylate . for this purpose , 50 μl of polymerization solution was spread into the void area and immediately covered with a hydrophobically silanized glass plate . the polymerization reaction proceeded for two hours at room temperature before the cover slide has been transferred into 10 ml water . the mask was easily removed from the cover slide leaving just the covalently bound gel pad at the void area . for the functionalization 20 μl of a 0 . 5 μm amino - modified reverse primer solution have been spread across the gel surface which was subsequently incubation in a humidity chamber for two hours at room temperature . the binding reaction was stopped by immersing the cover slide in blocking solution for 15 minutes . extensive rinsing with water removed all not bound reverse primers . the glass cover slide is the second sensor half element 2 , one surface which carries the reverse primer sensor compound 5 . 50 μl of a pcr mix which included a buffered system with 24 ng template dna , bovine serum albumin , trehalose , dntp mix , alexa cy3 fluorescently labeled dctp , ficoll and taq hotstar polymerase were applied to the first sensor half element 1 . ficoll is a water soluble polymer to self - seal a thin film fluidic reaction chamber at the contact with air . the second sensor half element 2 has been placed on top . the direct mechanical contact between gel pad and array has been enforced by gently squeezing out excess pcr mix . pcr cycling is started immediately afterwards and performed for 50 cycles of 95 ° c . for 30 sec , 62 ° c . for 30 sec and 70 ° c . for 2 min . separation of the surfaces of the two sensor half elements 1 , 2 is achieved by immersing the sensor array in a 2 × saline - sodium citrate buffer and 0 . 01 % sodium dodecyl sulfate washing solution for 10 minutes . the cover slide sensor half element 2 can be removed . several washing steps with buffer in stepwise dilutions and finally water dilute any remaining pcr mix components and unbound reaction mix from the surface of the sensor half element 1 . the glass slide 1 has been dried with pressurized air before scanning the surface with a genepix 4000b fluorescence scanner from axon . fluorescence values were recorded through 532 nm laser light excitation coupled with a cy3 optimized emission filter system . one representative sensor array section is shown in fig1 as black and white scan . it contains two rows of 6 spots each of the forward primer 4 , one row of the reverse primer 5 , the mixed primer combination 4 , 5 and the guide dots . the combination of the forward primer 4 from the first glass slide sensor half element 1 and the reverse primer 5 from the second sensor half element 2 yields the highest signals with more than 8500 counts . the background extension level is seen with around 500 counts at spots where the reverse primers 5 from both sensor half element surfaces 1 , 2 faced each other in the second row . the mixed primers display a combination of interfacial amplification of an effective half concentrated forward primer 4 and reverse primer 5 combination analogue rows 1 and 5 , and the contribution from the possible bridge amplification mechanism between the both primers 4 , 5 at the sensor half element 1 . eggers m . d ., hogan m . e . 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