in the sensor shown in fig1 an inert glass layer 1 separates an array of capacitive electrode pairs 3 , 3 ′, each connected to an inverting amplifier circuit 5 , from a conductive substance 6 whose equivalent circuit is indicated by the dotted - line schematic 7 . the electrode pairs 3 , 3 ′ are connected between the input and output of inverting amplifier 5 so as to yield a negative feedback . this sensor is similar to the capacitive distance sensor described in full detail in u . s . pat . no . 6 , 114 , 862 of tartagni et al ., which was developed for fingerprint identification . the non - contact biosensor of fig1 differs from that of the tartagni patent by the presence of a conductive material 6 at the inert layer 1 , which yields an equivalent circuit 7 comprising the virtual capacitors 8 , 9 , and 10 , connected by a resistor 12 , as indicated by dashed lines in fig1 . the impedances of different parts of substance 6 are measured by appropriate electronic interrogation of each pair of electrodes 3 , 3 ′, and each impedance value is assigned to an adjustable scale of 0 to 255 for display . with a single chip comprising an array of 256 × 364 electrode pairs , each 50 × 50 μm in size , the sensor can detect microscopic changes in impedance to about 10 - 20 μm from the outer surface of layer 1 , reducing interferences from substances or particles in a bulk solution . changes in the capacitance due to impedance changes at the outer surface of layer 1 are detected during interrogation of each capacitor pair 3 , 3 ′ at a selected alternating current frequency , e . g ., at 500 hz . such an array has the advantage of a known geometry and reproducibility . also , the electrical elements are isolated from the sample by the inert layer 1 , comprising layers of glass , such as phosphosilicate glass , silicon nitride , and / or silicon carbide . composite layers comprising a polymeric material , such as polyethylene , polypropylene , polymethacrylate polytetrafluoroethylene , or polycarbonate , may also be used . the isolation layer 1 also provides opportunities for the chemical attachment of active elements that can interact with biological molecules and particles . [ 0026 ] fig2 illustrates the grayscale change associated with kcl solutions of different concentration and hence different conductivity . the electric circuitry and software that services the array is similar to that disclosed in the afore - cited tartagni patent and displays the output of the sensor array as a grayscale image with a resolution of 1 : 256 . the grayscale image was calibrated by placing drops of kcl solution of different concentrations on the chip . images of 1 . 0 m , 0 . 001 m , and 0 . 0001 m kcl solutions are shown in fig2 . the sensor measures the size , shape , and impedance of the drop . the grayscale values can be used to image conductivity at high resolution without direct contact , potentially in such difficult systems as the external surfaces of live organisms or cultured cells . properties of physiological interest , such as concentrations of ions or molecular species , can be measured with this approach . for instance , urea can be sensed and measured by using bound urease to create nh 3 with subsequent reaction with hcl and conductivity change detection . the sensitiveness of the sensor - array chip of fig1 was tested with corn pollen dispersed over layer 1 . we mixed the pollen in a salt solution and thus obtained the results of fig3 . by comparing the computer images from the sensor array with pictures of microscope images one can see the very sensitive response of the chip , which is expressed by the gray scale . fig3 illustrates the grayscale identification of insulating bio - particles [ pollens ] in a conductive electrolyte . a comparison of optical and impedance images shows that both methods detect single particles or groups of particles . in order to assess some of the potential chemical and biochemical applications , imaging experiments were performed with 90 - μm corn pollen and 50 - μm pecan pollen particles . the imaging chip does not appear to respond to dry pollen , but if the particles are suspended in dilute phosphate buffer and a trace of surfactant , the particles can be imaged in contrast . fig3 shows optical and impedance images of corn pollen particles . the image of the corn pollen particles appeared from the suspension as it was drying . salt particles formed about each pollen particle , creating a dark region of low impedance against the lighter , high - impedance background . the large corn pollen particles overlap more than one electrode pair element , or pixel , so that statistical analysis of the two - dimensional data could be used to estimate the average particle size . this is confirmed by the impedance images of the grains in fig3 . [ 0029 ] fig4 illustrates an alternative visualization technique using the smaller (˜ 50 μm ) pecan pollen . addition of 1 % glycerol to the solution prevented complete drying and crystallization of the phosphate buffer , so that the background remained conductive ( dark ). the glycerol coated the particles making them non - conductive against the more conductive background . therefore , where the pollen particles displaced the evenly drying solvent , their images appear as lighter shades of gray , which is a negative of the technique used to produce fig3 . comparisons with optical images can be used for calibration and make the interpretation of the impedance images straightforward . in fig5 conductive carbon black particles ( vulcan xc - 72 ) are imaged after deposition from a low - conductivity nonionic detergent solution in which they were suspended . of course , these particles have very high conductivity and are imaged as the darker spots on the grayscale image . a simple chemical sensor was prepared by mixing conductive carbon black and ordinary silicone vacuum grease . a thin layer of this mixture was coated on the outer surface 1 of fig1 . the image of the smear of the mixture was initially light colored , in fact , lighter than the background grey value of the sensor , which implies that the thin coated layer was non - conductive . upon exposure to chloroform vapor , the image became considerably darker over a short time period , probably due to an increase in the number of conductive paths formed by the carbon particles caused by changes induced in the silicone - carbon layer upon solvent vapor exposure . this simple experiment demonstrates the potential of the capacitor array for chemical sensing applications . other materials are also expected to exhibit impedance pattern changes upon exposure to certain vapors , e . g , nafion with water vapor . the outer surface of the inert layer 1 can be chemically derivatized , and selectivity can be induced by the covalent binding of antibodies or oligonucleotides , using a procedure similar to that described in u . s . pat . no . 5 , 567 , 301 of stetter et al . and in the afore - cited publications by stetter et al . ( 1994 ) and feng et al . ( 1996 ). mammalian cells , bacterial spores , and some live microorganisms are large enough to be visualized by the array in its present form . quantitative measurements of ions in immediate proximity to cell membranes have been shown to be possible . coating with reactive compounds may enable selective gas sensing . because of the two - dimensional structure of the sensor , multiple channels of information can be acquired at one time . surface attachment of particles , such as pollen can be effectuated by activating the surface of layer 1 chemically and reacting with particles bearing amino groups on their surfaces . the chemically - attached particles should not be washed off or displaced by salt solutions , and should resist mild abrasion . this provides the basis for a generic type of biosensor that can be made selective to any type of bacteria , blood cell , or other biological particle by simply attaching an antibody that reacts with the particle of interest . to bring the binding phenomenon within the visible size range of the sensor array of fig1 one immunochemical detection method makes use of a conventional “ sandwich ” type of assay . it consists of chemically attaching an antibody to the surface 1 ; capturing bacteria on the prepared surface ; and reacting the surface with gold particles coated with more of the same antibody . the cells binding to the antibody on surface 1 may be smaller than the electrodes 3 , 3 ′ and therefore not large enough to be seen with the array . however , their sides farthest from surface 1 will have their antibody - reactive groups still available to react with more antibodies which are attached to larger gold particles . if these are large enough , they become visible to the array , since gold is electrically conductive . alternatively , an array of tinier pixels , e . g ., 10 μm × 10 μm or 1 μm × 1 μm in size , or a disposition of a derivatized surface pattern directly in the gaps between electrodes 3 and 3 ′ of each capacitive pair , may yield improved imaging and resolution of smaller particles . thus , by selectively derivatizing the outer surface of layer 1 to allow only selected biological particles , such as cells , spores , pollen grains or other specific cell lines , to attach thereto , it becomes possible to monitor the size , shape , viability , type , or status of selected biological particles . simultaneous imaging , recognition , and quantitation of two or more different analytes , i . e ., substances of interest , may be achieved by forming two or more differently derivatized surface layer patterns , each corresponding to a different analyte . such multiple derivatizations may be effectuated by known masking and dipping or spin - coating techniques , preferably utilizing photolithography to achieve maximum resolution and precision . each different pattern can be separately imaged by a programmed interrogation sequence . it is thus clear that impedance changes are useful in the detection of chemical and biological substances . the molecular basis is easy to understand even if only ph changes or water activity changes are taken into account . the configuration of fig1 which allows the sensing electrodes to be beneath the glass and yet the impedance near the glass surface to be controlling and thereby measured , can thus be seen to open the way to a broad range of chemical and biological sensing and imaging applications . various gases or vapors which are hazardous or emanate from hazardous or illicit substances , such as chemical warfare agents , carcinogenic or otherwise toxic industrial emissions products , explosive compounds , or narcotics may thus be detected and possibly quantitated . outstanding examples of such applications are observations of molecular or cellular parameters , such as the toxins , cell lines , viability or metabolic changes , or detection of cancer cells or of food - borne pathogens . by comparing the images of observed cells with a computerized data base of cancerous cell shapes or of food pathogen shapes , it becomes possible to diagnose cancer or food contamination at computer speeds . also , by following changes in impedance patterns , it becomes possible to perform biochemical or biological imaging in vivo on living tissue or on living cells or to monitor metabolic changes in real time or the progress of cryosurgery . there will now be obvious many variations and modifications of the afore - disclosed embodiments to persons skilled in the art , e . g ., changes in materials , in the shapes of the electrodes , in the insulating layer or the methods of electronic interrogation of the electrodes , which may yield improvements in sensitivity , selectivity , stability or response time . all of these variations and modifications will remain within the scope of this invention if defined by the following claims .
a unique imaging sensor consists of a two - dimensional array of impedance electrode elements separated from chemical or biological samples contained in or containing fluids by a fluid - impervious layer which prevents electrode fouling . it can serve for various chemical and biological sensing applications , ranging from diagnosing cancer to monitoring metabolic changes in real time or the progress of cryosurgery . the imaging can be applied to single cells in a manner that allows only specific cell lines to attach , then monitors their viability , type , and status with impedance , and further measures their size and shape by use the two - dimensionality of the electrodes array .