rocHENTeaL william R. Heineman an Departaent of university of a chenistry. cincinmaei brian Haleall cincinnati, ohio 45221-0172 apermact Biosensors paged on biological reaction that is very ‘specific for” the analyte” can. achieve a "high level “of Selectivity. Examples are given for sensors based “on enzymatic and immunological Feactions. hn ensynatic-based ‘ensyne ‘ntrappea ina. polyseric network of poly(vinyl elcohol) that be formed by ganna radiation crose-linking. Capillary immunoassays that require less than 100 ul of sample and that can detect extremely snail amounts “of analyte are examples of sensors Based on “immunological reactions. x detection linit of only 30,000 nolecules for Ygo in serux wae achieved with a eandvich assay | asing “a. TomuL henatocrit. blood collection tube as the capillary. mrnopuerrox one of the most difficult aspects of sensor development is the achievenent of Adequate. selectivity. for “the intended Application. one way to achieve selectivity As by means’ of @ biological reaction that is very specific for the target analyte: Fer hig reason, there is considerable interest in ‘biochemical sensors, or biosensors (1): The ‘response of a biosensor is based on a specific blolegicai reaction that is coupled £0 0a chenical sensor in order to inpart th selectivity of the biological reaction to the Gevice. Tis hae been accomplished by the Amnobilization of biological materials euch as” enzymes and. antibodies onto chemical Sensing elements such as electrodes, optical Fibers, and piezoelectric naterials. This paper describes sone of our researen on the incorporation of enzymes snd Antibodies as seneing elenents in blowensor ‘The enzyme work involves the investigation of ganna” radiation ae a. tecnnique for Imobitizing enzymes ‘on Sensor surfaces by 0-7803-0456-X/92 $3.00 © 1992 IEEE 102 + entrapnent in polymer networks. The antibody work involves the development of inmunoassays forthe analysis of very small samples. by coupling a capiiiary assay with electrochemical detection: XMMOBILIZATION OF ENZYMES ON ELECTRODES 1 POLYMER NETWORKS FORMED FORKED BY BY. GANMA ‘RADIATION CROSSLINKING Polymer filas inmobilized on electrode surfaces are an effective means of achieving Selectivity for an electrochesical sensor ‘The polymer can inpart selectivity by vir‘ of) one or a. combination” of several Rechanisns. (i) "specitic interactions between the polymer film and components in the sample can enhance the electrochemical signal for the analyte and. dininian it for interferences. "analyte can” selectively partition into the film by electrostatic, hydrogen” "bending, | nydrophabie, of coordination “interactions. (il) Restricted Giffusion into the polymer based on molecular Size can provide useful selectivity for Samples" in which interferences are. Larger than ‘the ‘analyse. (ili) The. polymer” ean participate indirectly in ‘the achievenent of Belectivicy by providing = matrix for. the immobilization of an enzyme ‘or an antibody that interacts with the analyte. Water-soluble polymers immobilized by ganna radiation have been investigated 28,3 neans of imparting selectivity to electrochemical “sensors by meana of the Sechenians qutlined above (2-9). canna rays Generate radicals in the polymer that cause Bolynerization and/or cross-linking of the ‘resulting polymer network “is insoiuble, yet ie will avell in water because of ite hydrophilic character.” ‘The svelling of ‘the network can be. controlled by the amount’ of mononer” mixed “with the polymer prior to cross-linking and the radiation Sosage. ‘Tho polymers" poly(W= vinyipyrrolidene) (PuvP}, poly(vinyl aicohal) (PVAL}, and poly (dinethyldialtylannonium chloride) TPOMDAAC) "have been Investigated for the purpose of developing electrochesicalSome advantages of this technique for preparing ‘polymer-nodified electrochenical Sensors are ‘iisted below. + simplicity procedure of the cross-linking + no removal of excess reactant (s) or by-product (2) + no "shadowing" + formation of # sterile device ‘he minimization of shadowing, Grose-linking by UY radiation, nigh penetration of ganna ray. comparea to is due to the Sone enzynes are sufficiently resistant to gamma radiation to be immobilized in a polymer matrix on an electrode. We have Benonstrated thie for glucose oxidase (6 alkaline phosphatace (8), and lactate oxidase (2), immobilized in “PVAL. PVAL Gan be [mobilized on electrode surfaces by gama radiation cross-linking to form an adherent flim that ie. sufficiently hydrophilic to avell when inmereed in water.” The swelling Gives ‘access to the immobilized enzyme by Solution species that are sufficiently small fo penetrate the polymer network. ~r), poly(vinyl alcohol), PVAL For example, lactate oxidase (100) has been ‘ inmobilized on platinizea graphite electrodes by sandwiching a thin enzyme layer between two layers of PVAL. The three layers: are individually © spin” coated on the electrode, which is then subjected to gama [etediation.”' "the ‘resulting sensor, responds jactate by ‘electrochemical oxidation of ‘generated by ‘enzyne-catalyzed eae ‘of lactate that diffuses into the network- ‘enzyme reaction: Lop lactate +0, ——~= pyruvate + tz0. electrode reaction: H,0, ——> 0, + aH + 267 The current is proportional to the Concentration of hydrogen peroxide at the @lectrode surface, ‘vnich is proportional to the concentration of lactate in eolution. “A Glagram of the sencor and the nechanisn for Fesponse is shovn below. a i platinum layer is ; W302 02 PVAL €£1m with Immobilized LOD lactate of this type show rapid (10 feady state) hydroanperonetric Fesponee to ‘aliquotes of lactate injected Into a stirred buffer solution, ‘The linear range of response is 26 4M to 1.7 uM with a Getection Limit of 13 um. Maximum response to lactate is obtained in the dosage range of 2-6 Mrad. Lover response “at lover dosages is attributed to ioeb of enzyne fron incomplete cross-linking of the polyner. Nonirradiated electrodes Give a goed Initial response to lactate, but fais “initial response deer replay as the polymer and enzyme gradually dissolve fron "the surface. Lover “response at high Aoeagen is ‘attrimuted to radiation damage to fhe enzyno. "Sone responce can be obtained for electrodes with dosages an high as 36 ‘the temperature dependence of response gives an optimum at s5°C compared to an Sptinun activity at 25°C for the free enzyme, Tnaicating stabilization by the radiation Immobilization process. "The sensors are stable for over’ 80 days when stored in dry form or in phosphate-acide olution at pM 7 and ‘Cenperature with continuous CAPILLARY IMMUNOASGAY WITH ELECTROCHEMICAL Derection ‘the combination of an electrochemical sensor with an. immunological reaction provides analytical, technique with excellent selectivity and detection linits (10,11). Electrochenical immunoassays with oth neterogensous [competitive and candwich} and homogeneous formate have been developed. These aceaye are based on labels that are Glectroactive or catalyze ‘the production of an’ electroactive reaction product. The latter label type can provide very low detection "linits ‘by! ‘virtue ‘of the amplification” feature of catalysis. Electrochemical sensore are used to detect electroactive labels or products of catalytic Feactions. 103Heterogeneous enzyne _inmunossseys in which “alkaline phosphatase ‘Is the enzyme Jebel have been Geveloped for the following analytes: Gipnactetoprotein (12). Gheophyliine (29) , ge (dete) , digoxin (27), Shaereeomucold (ie). Detectfon is based oh SMaline “phosphatase catalysis of the following substrate/product systems: "phenyl phosphace/phenol and praninopheny! PRosphate/praminophenol’” (29). "in, both Eyetens, product de detected by oxidation at Sabon electrede by either liquid Chromatography (LEC) oF flow. injection analysis (FIAEC) with electrochemical Gdtettion, “"Aneiboay te inmopilizea by passive adsorption to the eurface of plastic Eivettes “or Teovalent attachment to glass Beats or glace nicrocapillaries. In most Cases, “electrochemical immunoassays exhibit {Over detection limits or shorter assay tines than spectrocscopie assays due to the low detection limit for product. we have developed capillary Anmunoassays that require less then 100 ub of sample and ‘that can detect extrenely small Gnounte of "analyte (20). “The capillary {Meunoeessy methodology ie based ona email Gapiliary with antibody, hich selectively Sehcte with the analyte, immobilized on the Tnner) Wall. ‘Immunological and ‘enzyme anplification reactions are carried out Inbide ‘the tube, and” the product of the enzyne reaction is actected Glectrochenically. Advantages of this fechnique compared to other methodologies are very onall sample requirements (100 ul or less) + extremely amall detectable ancunts + fast assays + multiple aseaye on the sane cample Enzyme immunoasseys in the sandvich format “using alkaline, phosphatase as the Eneyne label have been demonstrated. in the assay procedure, sample ie injected into the Gapilisry; ansijte in the sample binds with the antibody and is thereby collected on the Inner “surtace of the capillary. Enzyne= beled secondary antibody ie then injected {nto the tube to determine how much analyte has bean captured on the curface. The enzyme Tabel ia alkaline phosphatase. in the final atep, substrate (praminophenyi phosphate) is {njected into. the capillary for a fixed ineabation tine after which the \enzyne~ Generates” ‘product (praminophenol) ie Geaneferrea into an electrochemical detection syeten for the quantification step. The sheyme catalyzed reaction is shovn below. ap Operon Ae wo + Opa 104 A schonatic representation of the assay Procedure is shown below. lab reagent ab Gapitiary witn [ab Tneide surface [Ab coated with ab |Ab xb xB aaaition of ap sample (5), fab:s capture t's ae by Ab on wall ig of capillary bes Rees add secondary antibody with ‘ehsyae label (absaP) which Feacte with § on surtace of Sapitiary Rossembeat > Beater lamar Be Lhenicany b:sinbear me io Produst P de detected by injection into flow injection analyele syster with a thin [ayer anperonatric detector. Detection is by dnigselon of P at a potential of 200 nV ve. Ryiagels | The electrode reaction is shown oo‘The smali-volune capillary combined with Signal-amplification feature of an ao acsay ha enabled extraordinarily iow detection levels to be achieved. "For ‘example, a detection Limit of ‘only 30,000 molecules’ for Tec in serum ae achieved witha sandwich assay using a 70-uL hematocrit blood collection’ tube” as the capiliary (20). The linear range vas four orders’ of magnitude and the assay vas Complete in 30 min. We are presently working foulower this detection lint by using a 10° ME capiiiary. “Even lover detectable anounte: Should be possible with -al and “100-nb, Capillaries." We are also working to expand Ehe application of the technique te "ene determination of “snaller molecules by the Sequential saturation or competitive formats Target ‘analytes are digoxin, which is important. clinically, and atrazine, which is avherbicide of envitonmental interest. The Capillary. innunoassay should be usefal for onitoring neonates, fron which large, blood Samples cannot be tsken ACKNOWLEDGEMENTS Financial support vas provided by ‘The Bdison "Sensor “Technology” Genter and the Environmental Protection Agency. (3) A.P.F, Turner, 1. 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