detection of hanta virus using a ne w miniaturized ... · detection of hanta virus using a ne w...

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detected amperometrically. The per-formance of the new biosensor devicewas evaluated by testing blind bloodsamples from 80 deer mice. The resultsobtained were compared to dataobtained using other independent tech-niques such as enzyme-linkedimmunosorbent assay, strip immunoblotassay, and data obtained from theCenters for Disease Control andPrevention. The overall time of analysiswas 22 minutes. Statistical analysis of thedata indicates that the device perform-ance improved the assay with no false-positive or false-negative analysesdetected.

INTRODUCTIONHantaviruses are cosmopolite anthropo-zoonosis considered to be an emergingdisease. Four pathogenic types forhumans and some of the Bunyaviridaefamily are hosted by rodents and havebeen isolated: the Sin Nombre virus(SNV), which is responsible for thesevere American respiratory form; theHantaan and Seoul viruses, which areresponsible for hemorrhagic fevers withrenal syndrome (HFRS) of severe-to-moderate expression in Asia and the

Vol. 7, No. 1, 2007 • The Journal of Applied Research

Detection of Hantavirus Using aNew Miniaturized BiosensorDeviceRavil A. Sitdikov, PhD*Ebtisam S. Wilkins, PhD*Terry Yates, PhD†

Brian Hjelle, MD, PhD‡

*Department of Chemical and Nuclear Engineering, University of New Mexico,Albuquerque, New Mexico†Department of Biology, University of New Mexico, Albuquerque, New Mexico‡ Department of Pathology, School of Medicine, University of New Mexico, Albuquerque, NewMexico

KEY WORDS: biosensor device, flow-through amperometric immunosensor,Sin Nombre virus, hantavirus antibodies

ABSTRACTA new smaller, automated electrochemi-cal immunosensor device for fast detec-tion of specific anti-hantavirusantibodies in mice blood has been made.This device has a manifold to reduce thevolume of analytes to allow precisemeasurement and to prevent the dilu-tion of the samples and immunochemi-cals. Manual simulation of the samplingsystem could eliminate false-positive orfalse-negative sample results. The assaytechnique was based on a flow-throughamperometric immunosensor. Highlydispersed carbon particles with immobi-lized recombinant Sin Nombre han-tavirus nucleocapsid protein served asan immunosorbent and as an immuno-electrode material. A sandwich schemeimmunoassay was used. Naphthol wasformed as a result of an enzymaticreduction of naphthyl phosphate in thepresence of an alkaline phosphataselabel, using a diethanolamine instead ofa bicarbonate buffer. The reaction was

The Journal of Applied Research • Vol. 7, No. 1, 2007 87

Balkans; and the Puumala virus, which isresponsible for HFRS of moderateexpression or the nephropathia epidemi-ca in Europe.1-3

To better understand the naturalhistory of this virus population, thedynamics and temporal pattern of infec-tion of its rodent hosts were studied insoutheastern Colorado from 1995 to2000. SNV is an etiologic agent of han-tavirus cardiopulmonary syndrome(HCPS). The presence of 2 hantaviruses,SNV in deer mice (Peromyscus manicu-latus) and El Moro Canyon virus inWestern harvest mice (Reithrodontomysmegalotis) were investigated at studysites.4-6

Hantavirus infections usually occurwhen humans inhale contaminatedrodent excreta.7,8 Wetting down deadrodents and their excreta reduces thechance that the virus will be airborne, aswell as kills the virus particles them-selves.9,10

Differences may exist between natu-rally infected rodents with respect toviral shedding, as illustrated when tissue

samples and excreta/secreta were testedfor viral DNA.11,12 Other saliva/oropha-ryngeal fluid and urine were collectedfrom seropositive animals, and the viralgenome was amplified for evidence ofSNV infection using deer mice fromsouthern Manitoba, Canada.

Enzyme-linked immunosorbentassay (ELISA) and Western blot testswere developed using a negative serumpanel and a blinded serum panel fromthe United States. They contained acute-phase sera from patients with HCPS andwere evaluated for the serological detec-tion of human infections caused by SNV,including those imported to Europe. Apolyhistidine-tagged recombinant nucle-ocapsid protein (RNP) of SNV wasexpressed in Saccharomyces cerevisiaeand purified by nickel chelation chro-matography. ELISA and the Westernblot tests were developed on the basis ofthe purified SNV RNP R-capture andindirect IgM and IgG. Based on thespecificity, the results were found to be100% for SNV R-capture and indirectIgM and IgG ELISAs.13-16

In southern Brazil, another com-plete sequenced S segment of hantaviruswas detected from 12 patients with han-tavirus pulmonary syndrome (HPS),which was diagnosed in different years,in distinct areas, and with a broad spec-trum of clinical signs. Identical S pro-teins of hantavirus from Paraná werefound despite these differences exceptfor 1 amino acid substitution.Phylogenetic analyses of the complete Ssegment nucleotide and amino acidsequences indicated that hantavirusesfrom Paraná form a distinct collectionfrom those circulating in South andNorth America.9,17,18

New Mexico had the greatest over-all incidence of hantavirus among thestates, followed by Montana.4,6 NewMexico also had the highest incidencebased on rural human population sizes,but Utah, Nevada, Montana, Arizona,

Figure 1. Photo of portable automatedimmunosensor device.

uted worldwide and are assumed toshare an extensive period of coevolutionwith the host. The degree of relatednessbetween virus serotypes normally coin-cides with the relatedness between theirrespective hosts. No known diseases areassociated with hantavirus infections inrodents, a fact that underlies the amica-ble relationship between virus and hostdeveloped by mutual interaction overhundreds of thousands of years.Antibodies against hantaviruses are alsopresent in domestic and wild animalssuch as house cats, dogs, pigs, cattle, anddeer. Domestic animals and rodents livejointly in a similar habitat, which wouldindicate that the transmission of han-taviruses from rodents to domestic ani-mals seems to be possible provided thatthe target organs, tissues, and cellparenchyma of the cohabitat domesticanimals possess adequate virus receptorsand are suitable for hantavirus entry andreplication.7,19,21

HFRS is a disease characterized byrenal failure, hemorrhage, and shock,and is predominantly caused by theserotypes Hantaan, Seoul, Puumala, and

Vol. 7, No. 1, 2007 • The Journal of Applied Research88

and Colorado, in that order, also report-ed high incidences.4,6 The host-viruscycle must be understood and additionaldata regarding the exact conditions ofhuman behaviors and exposure to theetiologic agent must be collected toallow a precise risk assessment ofacquiring HPS.19-21

Through July 6, 2005, a total of 396cases of HPS have been reported in theUnited States.4 The case count startedwhen the disease was recognized in May1993. When it was first recognized in the4 corners region of the United States,HPS had a lethality of more than 50%.Analyses of individual viruses indicatedthat the cause of HPS was a geneticallydistinct hantavirus, which was thereforetermed the Sin Nombre virus or SNV.The major causative agent of HCPS inNorth America is SNV carried by thedeer mouse. After a cluster of acute res-piratory distress syndrome deaths in thesouthwestern United States, HCPS wasrecognized. Thirty-six percent of allreported cases have resulted in death.2,19-21

Using specific rodent species astheir reservoirs, hantaviruses are distrib-

Figure 2. Cyclic voltammograph with 2 concentrations of 1-naphthol (AP product) in 0.1 Mdiethanolamine buffer solution: curve 1 = 1 mM; curve 2 = 2 mM; curve 3 = 1 mM of 1-naphthyl,and curve 4 = 0.1 M diethanolamine buffer solution.


Dobrava.9 Hantavirus isolates recentlyhave been identified and classified inpreviously unaffected regions includingNorth, Central, and South America.Outbreaks in the United States funda-mentally changed the knowledge aboutthe appearance of the hantavirus clinicalpicture, mortality, origin, and transmis-sion route in human beings. Numerousfactors influence the ability of apathogen to remain unnoticed or evolveinto a worldwide threat. Ever-changingenvironmental conditions and variationsin human behavior, including populationdevelopment, nutrition, education,social, and health status, are factors thataffect the correlation between an adapt-able pathogen such as hantavirus and itshost.2,7,8

Development of rapid, reliable, andsensitive immunoassay approachesrequires the application of serologicalmethods for the detection of hantavirusin the field or for epidemiological stud-ies. Clinical diagnosis of hantavirusinfection has been routinely confirmedby Western blot assays, hemagglutina-tion inhibition assays, immunofluores-cence antibody assay, or ELISA withnative viral antigens. A major disadvan-tage of these tests is that hantavirusesare highly pathogenic; handling themrequires Biosafety Level 3 (BSL-3) labo-ratory facilities. Therefore, these conven-tional immunoassay techniques can onlybe used in specially equipped hospitals

and laboratories, requiring highlytrained personnel. Analysis time of aconventional immunoassay is normally 6to 24 hours.16

An alternate approach to theimmunoassay is the flow-through tech-nique that uses electrochemical and flu-orescent detection methods. Primaryadvantages of electrochemical detectionmethods are their simplicity, selectivity,and sensitivity. An important advantageof flow immunoassay is the high area-to-volume ratio of solid to liquid phasesthat reduces transport limitations andrequires a significantly shorter analysistime.22,23

In this article the design and use ofa new, highly sensitive and specific flow-through portable device for rapid detec-tion of hantavirus infection thatovercomes the limitations associatedwith these conventional techniques isdiscussed. The immunosensor was basedon conducting ELISAs by using flow-through porous immunosorbents cou-pled with electrochemical transductionmechanisms. Schematic of the flow-through immunosensor assembly isdescribed in our previous publica-tions.24,25

ELECTROCHEMICAL BIOSENSORS In the University of New Mexico labora-tory, this sensor was tested for han-tavirus detection of rodent blood in thelaboratory as well as in the field. Other

Table 1. Conditions of the Oxidation of 3 Alkaline Phosphatase Products (2 mM) on a CarbonElectrode in the Different Buffer Media With pH 9.8

Maximum Oxidation Potential, mV Current Density, µA/mm2

Buffer Nitro NitroSolution* 1-Naphthol Phenol Phenol 1-Naphthol Phenol Phenol0.1M BcBS +200 ± 40 +410± 20 - 5.4 4.8 - 0.1M DEABS +220 ± 65 +470 ± 20 +850 ± 40 8.2 5.5 5.00.1M MEABS +210 ± 50 +540 ± 45 - 3.5 3.0 -

BcBS = bicarbonate buffer solution; DEABS = diethanolamine buffer solution; and MEABS = methylaminoethanolbuffer solution.


viruses, such as para influenza andinfluenza A viruses are also beingtested.26,27

MethodologyHighly dispersed carbon has a large sur-face area and, at the same time, func-tions as the working electrode becauseof its conductive nature. It has high areaper unit volume. This provided the basisfor immobilization of antibodies on thecarbon. This process augmented theproximity of the biological componentswith the transducer, a very essential fac-tor for biosensor development.23,24,26

ULTI (ultra low temperature isotropic)carbon was used as the material for theworking electrode in finely dispersedpowder form (a fraction smaller than 53-micron using sieve #270 accordingAmerican Society for Testing andMaterials Standards). Current collectionwas achieved through a carbon rod. TheAg/AgCl reference electrode and thecarbon counter electrode were prepared.Amperometry is a technique where theoutput of the sensor is current, which ismeasured by applying a constant poten-tial between the working and referenceelectrodes. The signal is caused by anelectrochemical process involving theanalyte, which takes place at the elec-trode’s surface. The potential differenceis termed working potential and is deter-mined by cyclic voltammetry.

The ULTI carbon modified by

immobilized recombinant replica of han-tavirus envelope was used for sandwichimmunoassay of hantavirus detec-tion.22,26 ULTI carbon, containing immo-bilized recombinant nucleocapsidprotein (RNP), was deposited in the dis-posable sensing element by vacuum. Thesmall size of the carbon particlesincreased the rate of immune interactionat each stage. Alkaline phosphatase(AP) was attached as a label to the anti-mouse IgG. AP catalyzed the hydrolysisof 1-naphthyl into 1-naphthol (Equation1). The scheme of reaction is as follows:

AP1-naphthyl phosphate + H2O !1-naph-thol + HPO4

-

Equation 1Electrochemical oxidation of 1-

naphthol formed the basis for determi-nation of the activity of the AP enzymeand quantification of the enzyme label.The amount of 1-naphthol formed bythis reaction, detected by electrochemi-cal oxidation, was a measure of theactivity of the AP label. Since theamount of antigen (analyte) determinesthe amount of AP-labeled antibodiesthat bind to form the sandwich, ampero-metric measurement of 1-naphtholformed was directly proportional to theanalyte concentration.

ImmunoassaysImmunoassays are based on the interac-tion of antigens and antibodies.

Table 2. A Sensor Noise and Response for 3 Buffers (pH 9.8) and 2 Substrates Used forAmperometric Detection of Alkaline Phosphatase Activity

Sensor Response at 10 ng/mLof a Native AP, µA1-Naphthyl Phenyl

Buffer* Noise, µA Phosphate Phosphate0.1 M BcBS -1.2 ± 0.03 0.8 ± 0.04 1.0 ± 0.1 0.1 M DEABS -0.6 ± 0.03 1.2 ± 0.04 1.1 ± 0.10.1 M MEABS -0.9 ± 0.07 0.9 ± 0.06 -

BcBS = bicarbonate buffer solution; DEABS = diethanolamine buffer solution; and MEABS = methylaminoethanolbuffer solution.


Antibodies are molecules of the immunesystem that perform a crucial role in thedefense mechanism. Antibodies exhibitstrong affinity for specified antigen mol-ecules, and their affinity for differentantigens differs in strength. Also thereare different determinants on the anti-gen molecules that elicit different recog-nition patterns with the antibodies.Antigens can be defined as moleculesthat are foreign to the body and findentry into the body. These can be chemi-cals or microorganisms like viruses andbacteria. Five main types (classes) ofimmunoglobulins can be identified inhuman beings and other mammals.28,29

The function of these antibodies is tobind to foreign molecules (antigens)entering the body in order to removethem. The removal is performed in asso-ciation with other components of theimmune system. Onset of immuneresponse of the body to an antigen ischaracterized by then production of alarge number of antibodies against theparticular antigen. The immune systemhas a large number of different antibod-ies (in the form of genetic information

inside the cells of the immune system)and is programmed to identify cells thatproduce most effective antibodiesagainst the particular antigen. Thus, theimmune system is capable of efficientlyprotecting the body from foreign mole-cules, cells, viruses, and bacteria.30,31

Antigen-Antibody InteractionsAntigen-antibody interactions are

governed by a simple equilibrium reac-tion as shown by Equation 2. The natureof this binding is noncovalent and isinfluenced by the pH, temperature, andionic strengths of the environment. Theequilibrium affinity constant (Kaff) is ameasure of the affinity of the binding(Equation 3).Ag + Ab " Ag-Ab Equation 2

Kaff = [Ag - Ab] / [Ag][Ab]Equation 3

In the above equations [Ag] refers toantigen concentration, [Ab] refers toantibody concentration, and [Ag - Ab]refers to the complexes formed by thebinding of antigens to antibodies. Kaff is

Figure 3. Calibration curve for 1-naphthol in 0.1 M diethanolamine buffer solution, pH 9.8.


the affinity constant and the inverse ofthis is called the dissociation constant(Kd). It should be noted that reaction 2is reversible. Such interactions can takeplace in a homogeneous medium or in aheterogeneous fashion, where one of theinteracting species is bound to somesolid phase. This molecular recognitionprocess is highly specific and leads togreat selectivity. The reaction does notproceed according to stoichiometricfashion due to the various valences ofthe antibodies and antigens. Antigenscan be proteins, polypeptides, or nonpro-tein molecules like carbohydrates. Small,low molecular weight molecules called“haptens” can also induce immuneresponse. Interactions with multivalentantigens lead to formation of complexesof immunospecies.

MATERIALS AND METHODS Reagents and MaterialsThe RNP of the hantavirus envelopewas obtained from Dr. B. Hjelle’s labo-ratory.15 Woodward’s reagent K

(N-ethyl-5-phenylisoxazolium-3!sul-fonate), trypsin inhibitor, 1-naphthylphosphatase, 1-naphthol, ethylenedi-aminetetraacetic acid, bovine serumalbumin (BSA), nitro blue tetrazoliumand Tween 20 were obtained from SigmaChemical Co. (St. Louis, MO).Diethanolamine and methylamino-ethanol were obtained from FlukaChemie GmbH (Buchs, Germany). AP-labeled anti-peromyscus leucopus(white-footed mouse), IgG (developedin goat), and para-nitrophenylphosphate(pNPP) were obtained from Kirkegaard& Perry Laboratories Inc.(Gaithersburg, MD).

Hantavirus positive, weak-positive,and negative (controls) deer mice bloodsamples were obtained from clinicaldiagnostic specimens at the Universityof New Mexico Health Sciences Center’sHantavirus Diagnostic Unit. Ultrafree-MC with 0.22-µm pore size was obtainedfrom Millipore Co. (Bedford, MA).ULTI carbon microdispersed powderwas provided free of charge by

Figure 4. Influence of the sensor’s background current drift to reliability of analytical signalsmeasurements. Points 1, 2, and 3 are analytical signals for samples with high, medium, and lowanalyte concentration, respectively; 5 is initial buffer current, and 6 is the final buffer current.


CarboMedics Inc (Austin, TX).

Immobilization TechniquesWoodward’s reagent K immobilization isa technique for obtaining covalent link-age of the proteins to the surface of thecarbon (covalently linked imunoagent-solid phase conjugates). First, an activa-tion of the carboxylic group of thecarbon solid support was performed.Second, coupling of the proteins to theactivated solid support occurred. The pHof the solution with Woodward’s reagentK (20 mg/mL) in water was adjusted to4.5 using diluted NaOH solution, fol-lowed by suspension of 10 mg of ULTIcarbon (size of the carbon particles was<53 µm) in 2 mL of Woodward’s reagentK solution. This was followed by incuba-tion at room temperature for 2 hourswith shaking. The suspension was laterwashed 5 times with distilled water byrepeated centrifugation and removal ofthe supernatant. Carbon thus treatedwith Woodward’s reagent K was sus-pended in 2 mL of a solution of han-tavirus RNP (20 µg/mL) in 0.02 M

Na-phosphate buffer solution (PBS) pH7.8, containing 0.15 M NaCl. The suspen-sion was incubated at room temperaturefor 2 hours with shaking. The suspensionwas later washed 5 times with PBS byrepeated centrifugation and removal ofthe supernatant. After incubation, 5mg/mL solution of trypsin inhibitor inPBS was added to the same suspensionas a blocking agent and incubated for anadditional 2 hours at room temperaturewith shaking. The suspension was finallywashed 5 times with PBS by repeatedcentrifugation (5 minutes each) andremoval of the supernatant. Theimmunosorbent was stored in the samebuffer solution at 4°C.

Hantavirus AssayThe detection of SNV infection wasbased on the determination of antibod-ies against the SNV in blood samples.The disposable sensing element wasplaced into the holder and firmly fixedin the immunocolumn, having intimatecontact with the immunosorbent.26 Then,the solutions were pumped through the

Figure 5. ELISA analyses using mouse blood controls at varying dilutions. ELISA = enzyme-linkedimmunosorbent assay. OD405 = optical density at 405 nm.


Figure 6B. Estimation of hantavirus mice blood controls assay by amperometric (immunosensor)and optical (ELISA reader) detection. 0.5 mg of anti-hantavirus ULTI carbon immunosorbent wasused for both of cases. ULTI = ultra low temperature isotropic; ELISA = enzyme-linked immunosor-bent assay.

Figure 6A. Comparison of ULTI carbon and standard ELISA polystyrene microplate immunosor-bents for hantavirus mice blood controls estimation. The optical detection of alkaline phos-phatase-labeled activity was used for both of cases. ULTI = ultra low temperature isotropic; ELISA= enzyme-linked immunosorbent assay.


immunosensor. The flow rate of thereagents through the immunocolumn inthe device was 120 µL/min.

The assay involved a sandwichscheme of detection in the followingorder:

Prewashing: 0.02 M Na-phosphatebuffer solution (pH 7.8) containing 0.15M NaCl and 0.1% Tween 20 (Na-phos-phate buffer solution with tween -PBST) flowed through the immunoelec-trode for 1 minute.

Sample injection: blood samplediluted to 500 times by PBST, tested foranti-hantavirus antibodies (target ana-lyte), flowed for 3 minutes. The targetanalyte was bound to the surface of theimmunoelectrode.

Washing: the PBST flowed for 3minutes.

Conjugate injection: 1 µg/mL of AP-labeled anti-mouse IgG (conjugate)against the target analyte flowedthrough the immunocolumn for 8 min-utes.

Washing: the diethanolamine buffersolution (DEABS) prepared as follows:0.1 M diethanolamine (pH 9.8), contain-ing 0.15 M NaCl and 1 mg/mL MgCl2,flowed through the immunocolumn for 4minutes.

Substrate injection and amperomet-ric measurement: 0.1 M diethanolamine(pH 9.8), containing 0.15 M NaCl, 1mg/mL MgCl2, and 1 mM #-naphthylphosphate flowed through the immuno-electrode for 4 minutes. The amperomet-ric measurements were performed at afixed electrode potential (+250 mV ver-sus Ag/AgCl).

ELISA Standard ELISA of Mice BloodSamplesThe ELISA of mice blood control sam-ples was performed by standard sand-wich scheme.

One hundred microliters of the han-tavirus RNP solution (8.5 µg/mL) in 0.02

Table 3. Correlation Among Electrochemical Immunosensor Device, ELISA, and StripImmunoblot Assay Data for Anti-Hantavirus Antibodies Assay of Mice Blood Samples

Sample, Immunosensor Device Standard ELISA SIABlood Name i, µA Result OD405 Result Data*Negative (-) 0.26 Control 0.128 Control - Weak-positive (+) 0.75 Control 0.250 Control -

1 0.19 Negative 0.126 Negative Negative 2 2.02 Positive 0.858 Positive Positive 3 0.32 Negative 0.130 Negative Negative4 2.12 Positive 2.360 Positive Positive5 0.19 Negative 0.131 Negative Negative6 2.54 Positive 1.353 Positive Positive7 0.29 Negative 0.130 Negative Negative8 0.18 Negative 0.129 Negative Negative9 0.23 Negative 0.128 Negative Negative10 0.26 Negative 0.132 Negative Negative

*The Strip Immunoblot Assay data were presented by Dr. B. Hjelle’s laboratory (Center for Infectious Diseases andImmunity, University of New Mexico Health Sciences Center).


Table 4. Correlation of Anti-Hantavirus Antibodies Assay of Mice Blood Samples BetweenElectrochemical Immunosensor Device Results and CDC Data*

StandardSample, System,† Device, Result of CDCBlood Name microamperes microamperes Assay DataNegative (-) 0.15 0.26 Control -Weak-positive (+) 0.95 0.75 Control -MF 1 0.18 0.18 (-) (-)MF 2 0.22 0.25 (-) (-)MF 3 0.30 0.28 (-) (-)MF 4 1.55 1.70 (+) (+)MF 5 0.45 0.54 (-) (-)MF 6 0.35 0.42 (-) (-)MF 7 0.25 0.30 (-) (-)MF 8 0.28 0.30 (-) (-)MF 9 0.25 0.21 (-) (-)MF 10 1.60 1.30 (+) (+)MF 11 0.22 0.18 (-) (-)MF 12 0.35 0.22 (-) (-)MF 13 0.20 0.32 (-) (-)MF 14 0.15 0.37 (-) (-)MF 15 0.30 0.41 (-) (-)MF 16 2.60 2.75 (+) (+)MF 17 0.35 0.21 (-) (-)MF 18 0.32 0.23 (-) (-)MF 19 0.32 0.30 (-) (-)MF 20 0.25 0.28 (-) (-)MF 21 0.20 0.30 (-) (-)MF 22 0.35 0.33 (-) (-)MF 23 0.30 0.25 (-) (-)MF 24 0.18 0.16 (-) (-)MF 25 0.35 0.26 (-) (-)MF 26 0.47 0.39 (-) (-)MF 27 0.25 0.26 (-) (-)MF 28 0.25 0.30 (-) (-)MF 29 1.30 0.97 (+) (+)MF 30 0.20 0.32 (-) (-)MF 32 0.35 0.26 (-) (-)MF 33 0.22 0.15 (-) (-)MF 34 0.30 0.12 (-) (-)MF 35 0.25 0.28 (-) (-)MF 36 2.80 2.72 (+) (+)MF 37 0.11 0.43 (-) (-)MF 38 0.35 0.26 (-) (-)MF 41 0.30 0.24 (-) (-)


StandardSample, System,† Device, Result of CDCBlood Name microamperes microamperes Assay DataMF 42 0.25 0.16 (-) (-)MF 43 0.30 0.21 (-) (-)MF 44 0.30 0.25 (-) (-)MF 45 0.35 0.17 (-) (-)MF 46 0.20 0.28 (-) (-)MF 47 0.16 0.20 (-) (-)MF 48 0.40 0.26 (-) (-)MF 49 0.30 0.24 (-) (-)MF 50 0.36 0.37 (-) (-)MF 51 1.80 2.07 (+) (+)MF 52 0.85 0.34 (-) (-)MF 53 2.10 1.35 (+) (+)MF 54 1.60 1.30 (+) (+)MF 55 0.20 0.29 (-) (-)MF 56 0.25 0.16 (-) (-)MF 57 0.30 0.22 (-) (-)MF 58 0.40 0.37 (-) (-)MF 59 0.35 0.29 (-) (-)MF 60 ? 0.30 (-) (-)MF 61 0.45 0.27 (-) (-)MF 62 0.35 0.14 (-) (-)MF 63 0.30 0.28 (-) (-)MF 64 0.40 0.28 (-) (-)MF 65 0.25 0.18 (-) (-)MF 66 1.60 1.28 (+) (+)MF 67 0.35 0.25 (-) (-)MF 68 0.35 0.35 (-) (-)MF 69 0.20 0.16 (-) (-)MF 70 0.40 0.35 (-) (-)MF 71 0.35 0.39 (-) (-)MF 73 0.83 0.20 (-) (-)MF 74 0.20 0.22 (-) (-)Human blood 0.15 0.15 Nonspecific -

* Deer mice blind blood samples and US CDC data were provide by Dr. Terry Yates (Museum of SouthwesternBiology, Department of Biology, University of New Mexico).†As the standard system, the potentiostat and X-Y recorder were used for registration of sensor’samperometric responses.

Table 4. Continued


M Na-phosphate buffer pH 7.8 contain-ing 0.15 M NaCl (PBS) were placed intothe wells of the ELISA plate and storedovernight at 4°C. The ELISA plate waswashed 3 times by adding 200 µL of PBSwith 0.1% Tween 20 (PBST) into eachwell. This procedure was then followedby the addition of 150 µL of a solutionof 2% bovine serum albumin (BSA) inPBST, as a blocking agent, with incuba-tion at room temperature for 30 min-utes. The wells were then washed 3 timeswith PBST.

Then the ELISA plate was incubat-ed with mice blood samples diluted from200 to 4000 times in PBST. The PBSTwas used as a blank control. The platewas incubated at room temperature for2 hours. The wells were then washed 3times with PBST. Goat anti-mouse AP-labeled conjugate was prepared by dilut-

ing the commercially obtained solutionto a ratio of 1:1000 in PBST, and then100 µL of the diluted conjugate solutionwas added into each of the wells. Thiswas followed by incubation of the plateat room temperature for 2 hours. Later,the wells were washed 3 times withPBST and once with DEABS. Finally100 µL of DEABS containing 1 mg/mLof pNPP AP substrate was added toeach of the wells. After 5 or 10 minutes,100 µL of the 5% EDTA solution wasadded into each of the wells as a stopreagent. Results of the ELISA wereobtained by reading the optical densityat 405 nm (E-312e Bio-kinetic reader,Bio-Tech Instruments, IL).

ELISA Control of the Anti-HantavirusULTI Carbon Immunosorbent

As a parallel method for control of

Figure 7. Linear regression for anti-hantavirus antibody assay data obtained by ELISA andimmunosensor device. ELISA = enzyme-linked immunosorbent assay.! Strong-positive mouse blood control! Weak-positive mouse blood control! Negative mouse blood control" Blood samples


the immunospecificity of ULTI carbonimmunosorbent, a modified ELISA wasused. This experiment was performed atroom temperature.

Carbon immunosorbent (0.5 mg)was placed into the each microcentrifu-gal filter unit (0.22 µm pore size) andcentrifuged for 5 minutes. Filter unitswith adsorbed immunosorbent werewashed by 400 µL of PBST and cen-trifuged for 3 minutes. This procedurewas followed by the addition of 250 µLof a 1:500 diluted mice blood sample inPBST into each filter and incubated for20 minutes with vortex shaking. The fil-ter units with immunosorbent were thencentrifuged and washed 3 times with

PBST. This was followed by the additionof 400 µL of 0.5 µg/mL AP conjugatesolution into each filter, which was incu-bated for 20 minutes with shaking. Afterthat, filter units were centrifuged andwashed 3 times as described previouslyand once by DEABS. This procedurewas then followed by the addition of 300µL of 1 mg/mL pNPP solution inDEABS and then incubated for 10 min-utes. After centrifuging the enzymaticproduct, colored solution from each fil-ter unit was collected and added (for200 µL) to wells of empty standardELISA plates. Results of this experi-ment were obtained by reading the opti-cal density at 405 nm.

Figure 8. Linear regression for anti-hantavirus antibody assay data (80 deer mice blood samples)obtained by immunosensor device and standard registration system." Weak-positive mouse blood control" Negative mouse blood control! Blood samplesP = positive and N = negative quadrants.


The Strip Immunoblot AssayThe strip immunoblot assay (SIA) wasprepared by subjecting affinity-purifiedSNV RNP antigen, along with serum-control intensity markers, to adsorptiononto nitrocellulose membranes by vacu-um. The membrane was then shreddedlengthwise into 1.6-x 100-mm stripsusing a hand-held paper shredder. Eachstrip was exposed to a 1:200 dilution ofdeer mouse serum for 4 to 16 hours, fol-lowed by washing in a detergent bufferand exposure to a 1:1000 dilution of ananti-peromyscus leucopus (white-footedmouse) IgG- AP conjugate. After wash-ing, the presence of membrane-boundAP was detected by exposing the stripsto nitro blue tetrazolium substrate. Thetest was considered positive if there wasany darkening of the RNP antigen bandon the strip. The serum-control lanesallow one to verify that the vacuum stepand the steps involving the use of theconjugated-antibody functioned asexpected; another extra band consists ofa small amount of an essentially inertdye, Coomassie brilliant blue, whichenables the reader to verify the correctorientation of the strip.32

The Cyclic Voltammetry of APEnzymatic Substrates and ProductsThe cyclic voltammetry apparatus con-sisted of a BAS CV-1 voltammograph(Bioanalytical Systems Inc., WestLafayette, IN), a BK precision model2832 digital multimeter (DynascanCorporation, Chicago, IL), a HoustonInstrument model 100 X-Y recorder(Houston Instruments, Houston, TX),home-made glass electrochemical cell.

The electrochemical cell with 10 mL ofelectrolyte volume included a carbonworking electrode (4.5 mm diameter), acarbon counter electrode, an Ag/AgClreference electrode, and intensive mag-net stirring system. The scan rate poten-tial was 20 mV/sec. The results of theseexperiments allowed choosing thepotentials of working electrode foramperometric detection of products forAP-labeled detection.

RESULTS AND DISCUSSIONThe detection of SNV infection wasbased on the determination of antibod-ies against the virus in hemolyzed miceblood.26,27 The SNV RNP was used as apotential diagnostic antigen for the pur-pose of developing a rapid and reliableelectrochemical immunosensor fordetection of anti-SNV antibodies.22 Theexperiments were conducted with miceblood containing antibodies againstSNV caused by infection with SNV. Thespecificity of the test was determined byusing a negative, weak-positive, andstrong-positive control blood (miceblood with no anti-SNV antibodies, withlow-level and with high-level anti-SNVantibodies). The hantavirus-specificresponses of the sample, as determinedwith an amperometric immunosensorwere compared with the levels detectedby ELISA.

In this experiment, the SIA basedon the SNV RNP was used as a refer-ence method for identification of thepositive and negative blood samples.Weak and strong-positive controls wereprepared by using mice that had beenonly recently infected (within the first 3

Table 5. Summary of the 70 Deer Mice Blood Samples Testing Data

Testing Total False- False-Method Samples Negative Positive Negative Positive New Device 70 61 9 0 0CDC Results 70 61 9 0 0


weeks) for the former and mice that hadbeen inoculated with virus in the moredistant past for the latter. A mixed selec-tion of deer mice blood samples, includ-ing some from mice that had beenexperimentally infected with SNV wasused. The experimental infection modelfor SNV in the deer mouse was provid-ed for testing by other methods in ablinded manner.19

Sensor Design The immunoassay system design conceptwas based on conducting ELISAs byusing flow of analyte and immunochemi-cals through porous immunosorbents(carbon) coupled with electrochemical-based transduction mechanisms as ameans of overcoming the limitationsassociated with conventional techniques.From an engineering perspective, theuse of flow-based systems allowed foreasier automation of the analysis proce-dure because the flow was usually driv-en by pumps and valves that were easilycontrolled by microprocessors.25

Description of the Total System The automated immunoassay device(Figure 1) was a set of electronics thatdrove an immunoelectrode and tested asample for anti-hantavirus antibodies.The device consisted of 4 blocks: apower supply, a potentiostat, a pumpingsystem, and a microcontroller. Thepower supply used either batteries orAC to generate all the voltages used inthe device. The potentiostat was used to

bias the immunoelectrode and to con-vert input current into a voltage signal.The 5-valve manifold and pumping sys-tem delivered the reagents used to testthe sample. The microcontroller ran theprogram used to test the sample andshowed the results on the liquid crystaldisplay.

Optimization of the AP-LabeledDetectionThe parameters (such as the flow rate ofthe reagents, the pH of conjugate andsubstrate solutions and their concentra-tions, and incubation times) were inves-tigated and optimized earlier.22,24,26 Usingthese conditions, promising results wereobtained during the application of thedevice in detecting antibodies againsthantavirus mice blood samples.However, several parameters have beenchanged. The parameters affecting boththe immunological reaction stage andthe amperometric transduction stage,namely working potential, nature of sub-strate, and buffer solutions, were investi-gated. Optimization of theelectrochemical detection was based onthe following criteria: maximum enzy-matic activity of the AP enzyme, maxi-mum sensor response (current density),and reproducibility of the sensorresponse evaluation.

Selection of a Working ElectrodePotential and Buffer Media forAmperometric Detection of AP Activity The working potential is the potential at

Table 6. Summary of the 10 Deer Mice Blood Samples Testing Data

Testing Total False- False-Method Samples Negative Positive Negative Positive New device 10 7 3 0 0Strip immunoblot 10 7 3 0 0assay

ELISA 10 7 3 0 0

ELISA = enzyme-linked immunosorbent assay.


which the working electrode is biasedwith respect to the reference electrode.A suitable working potential has to beselected in order to achieve efficientproduct formation during the enzymaticreaction of the conjugate. Selection of asuitable working potential was per-formed using cyclic voltammetry. Severalsubstrates have been reported for usewith AP label in electrochemicalimmunoassay.33-35 For amperometricdetection of AP activity, the workingvoltammograms were obtained in thepresence of 1 mM 1-naphthyl phosphate,phenyl phosphate, and pNPP as sub-strates of AP and its products in differ-ent buffer solutions with pH 9.8. Theregion of the oxidation current of 1-naphthol as an AP enzymatic reactionproduct was observed at a potentialbetween +100 and +400 mV (shown inFigure 2 as a red arrow for peaks curves1 and 2) for different buffer solutions.This range was used to conduct all of theexperiments.

Different buffer solutions and enzy-matic substrates were used to optimizethe current density and maximize oxida-tion potential for selection of the opti-mum conditions for AP detection asshown in Table 1.

Using the 3 buffer solutions (bicar-bonate, DEABS, and methylamino-ethanol buffer solution [MEABS]) and 3products (1-naphthol, phenol, nitro phe-nol), a potential was selected between+150 to +300 mV for the operating con-ditions of the experiments (Figure 2 andTable 1). The optimized range of oxida-tion potential was either +250 mV or+300 mV.

In order to evaluate the sensorresponse by maximizing the signal-to-noise ratio using +250 mV as potentialand 3 buffers solutions and 2 substrates,the optimum condition for the best sig-nal was DEABS, which had the lowestnoise and maximum sensitivity (Table2). Hence, DEABS and +250 mV as

potential were selected as the optimumconditions for amperometric registrationof 1-naphthol.

A calibration curve for the differentconcentrations of 1-naphthol in mM ver-sus the sensor signal in microamperes isshown in Figure 3. The minimum detec-tion limit of 1-naphthol is about 2 x 10-6

M. The data from this study fell in therange above 2 x 10-6 M.

Evaluation of the Analytical Signal ofFlow Amperometric SensorIn most flow-through electrochemicalsensors, the background current, whichis the baseline signal value, is veryimportant for evaluation of the controlsamples. A drift of the baseline signalcould become a source of low repro-ducibility of the data. The analytical sig-nal described in this immunosensordevice was the peak of the signal as itreached a steady-state current. Thevalue in microamperes of this peak cor-responded to the concentration of 1-naphthol, which is an AP enzymaticproduct. Figure 4 clearly shows the influ-ence of the baseline drift (backgroundcurrent) on the signal of different sam-ples. For example, sample 1 represents astrong positive, sample 2 is a weak-posi-tive, and sample 3 is a negative.

In some cases, the background cur-rent obtained by the device (end stage 5point in Figure 4) could be higher thancurrent for negative blood samples(point 3 in the same figure) or about thesame value for a weak-positive sample(point 2). The information about thebackground current drift during themeasurement stage was programmed forautomatic subtraction of the steady-statesignal of the sample from its steady-statebackground current and was used ascorrectional factor.

Detection of Hantavirus Antibodies inMice Blood SamplesSeventy blind blood samples from


hemolyzed deer mice were obtainedfrom the Museum of SouthwesternBiology, Department of Biology,University of New Mexico (Dr. Yates’sLaboratory). Positive, weak-positive, andnegative (controls) samples plus anadditional 10 deer mouse blind bloodsamples were obtained from clinicaldiagnostic specimens from the Centerfor Infectious Diseases and Immunity atthe University of New Mexico HealthSciences Center (Dr. Hielle). The resultsof these blind sensor tests were com-pared with the results from SIA andELISA techniques in addition to theELISA data obtained from the Centersfor Disease Control and Prevention(CDC) for the same 70 blood samples.Correlation of the data from theimmunosensor device and the signalsfrom the sensor, which were detected bya standard potentiostat and X-Yrecorder, were investigated.

ELISA Experiment with DifferentMouse Hantavirus Blood ControlsIn recent years, ELISA using poly-styrene plates has been shown to be aneffective tool for the detection of vari-ous analytes. ELISA is good tool for lab-oratory pre-control of differentimmunoreagents, such as antibodies,antigens, enzyme conjugates and theirimmunological and/or enzymatic activi-ty. In this investigation, ELISA was cho-sen as a standard technique forpreliminary estimation of the propertiesfor recombinant hantavirus NP, miceblood controls, and anti-mouse AP con-jugate.

Results of the sandwich ELISA fordifferent dilutions of mice blood con-trols from 1:200 dilution to 1:4000 dilu-tion are shown in Figure 5. These resultsreflect good range from 1:400 to 1:500dilutions. For these experiments, a dilu-tion of 1:500 was used; this is similar toprevious work,22 and it is recommendedto use this same dilution for future sam-

ples. The reason for that is that the dilu-tion ratio 1:200 showed similar strongdifferences between, positive, weak-posi-tive and negative samples using thesame immunoreagent.

In this case the ratios of optical den-sities, as analytical signals for ELISA,for strong-positive, weak-positive, andnegative hantavirus blood controls areas follows:strong-positive/negative = 5.2strong-positive/weak-positive = 4.3and weak-positive/negative = 1.4.

A dilution of 1:500 with only 0.72µL of each blood sample was sufficientfor a hantavirus assay by amperometricimmunosensor.

Electrochemical and Optical (ELISA)Control of ULTI Carbon Anti-Hantavirus ImmunosorbentThe aim of this experiment was to useELISA as a preliminary control of anti-hantavirus immunosorbent immunologicactivity. ULTI carbon particles withimmobilized hantavirus RNP were usedfor this experiment with mouse bloodcontrols. The amount of bounded immu-noenzyme complex onto the carbonimmunosorbent surface was estimatedby optical detection of AP-labeled activ-ity as described in “ELISA Control ofthe Anti-Hantavirus ULTI CarbonImmunosorbent” section. Results ofELISA for immunosorbent with 1:500times diluted mice blood controls areshown in Figure 6A. This was based onFigure 5, and the results from ELISAwith blood samples from hantavirus-pos-itive and hantavirus-negative mice. Inthis case, the ratios of the optical densi-ties for the 3 hantavirus blood controlswith carbon immunosorbent are as fol-lows:strong-positive/negative = 7.2strong-positive/weak-positive = 4.4and weak-positive/negative = 1.65.

A carbon immunosorbent was cho-sen because it had higher activity due to


the large surface area and/or specificity(strong-positive/negative = 7.2 andweak-positive/negative = 1.65). It wasdiscovered that the anti-hantavirus RNPthat was used in this experiment onpolystyrene ELISA microplates hadlower ratios (strong-positive/negative =5.2 and weak-positive/negative = 1.4)than did the carbon immunosorbent.Therefore, carbon anti-hantavirusimmunosorbent can be used more suc-cessfully than ELISA with higher sensi-tivity for detection of anti-hantavirusantibodies. Figure 6B shows that amper-ometric detection had greater sensitivityfor mice blood controls estimation thanthe optical method. Although the y-axisis not the same scale for both theimmunosensor and ELISA, the 2 detec-tion methods were combined in order tocompare the data from both methods.

Comparison of Immunosensor DeviceData with ELISA and SIA Table 3 shows that the results obtainedby the newly developed immunosensordevice used for assaying the 10 miceblood samples were similar to dataobtained by ELISA and SIA.

The same similarity was observedfor the additional 70 blood samples inwhich data were obtained from theCDC independently, see Table 4.

STATISTICAL ANALYSIS ANDDISCUSSIONLinear regression for anti-hantavirusantibody assay data obtained by ELISAand immunosensor device for 10 miceblood samples is shown in Figure 7. Thecorrelation equation for ELISA andimmunosensor device methods is Y =0.205 + 1.104X with regression coeffi-cient r=0.87194. Figure 7 illustrates thatthere is a good agreement betweenstrong-positive mouse blood dataobtained using the immunosensor deviceand ELISA. It also shows that there is asimilar correlation between negative

mouse blood data. The figure also showsthe 2 control samples for negative andweak-positive blood. The strong-positivesample had a much higher signal thanthe weak-positive control for the device,and was confirmed by ELISA.

The linear regression for all han-tavirus assay data (80 deer mice bloodsamples) obtained by immunosensordevice and standard cyclic voltammetrysystem are shown in Figure 8. Data fromstandard cyclic voltammetry commercialpotentiostat system in comparison withthe immunosensor device were obtainedusing the same disposable immunosen-sor column and an X-Y recorder. Thecorrelation equation for standard cyclicvoltammetry and the immunosensordevice was a liner regression describedby the following equation Y = 0.0079 +0.88X with regression coefficientr=0.9521. There was a good correlationbetween the standard cyclic voltamme-try and the immunosensor device. InFigure 8, the top right-hand quadrant ofthe graph illustrates the positive bloodsamples for both methods. The bottomleft-hand quadrant shows the negativeblood samples. The other 2 empty quad-rants show that there were no false-posi-tive or false-negative blood sampleresults when the standard cyclic voltam-metry or the immunosensor device wasused.

Finally all mice blood immunoassaydata obtained by the new immunosensordevice, CDC, ELISA, and SIA are pre-sented in Tables 5 and 6.

Seventy-one blind samples wereprovided by Dr. Terry Yates (Museum ofSouthwestern Biology, Department ofBiology, University of New Mexico). Thedata were confirmed by the CDC.Similar results were obtained using theimmunosensor device, and these were intotal agreement with the CDC data.Confirmation of the data obtained usingthe immunosensor device is obvious—they had no false-positive or false-nega-


tive results and were in agreement withthe CDC results. Detection using theimmunosensor device indicated that thissystem was in good agreement withstandard methods such as ELISA orstandard cyclic voltammetry.

Table 6 shows similar results from10 blind blood samples obtained fromthe Department of Pathology, School ofMedicine, University of New Mexico.Samples were tested using theimmunosensor device, ELISA per-formed by the authors, and SIA per-formed by Dr. B. Hjelle. As seen in Table6, all methods used were in good agree-ment with no false-positive or false-neg-ative results.

Analytical sensitivity is defined asthe change in signal relative to thechange in concentration of the analyte.All immunoassays suffer to some degreefrom the disadvantages of false-positivesand false-negatives. This influences thesensitivity and the specificity of amethod. Sensitivity of a device is its abil-ity to detect a particular species in asample solution, while specificity is itsability to detect nothing except thespecies. Sensitivity and specificity are the2 vital criteria that determine the per-formance of an analytical instrument.36-38

Sensitivity and the specificity of themethod were evaluated based onInternational Union of Pure andApplied Chemistry recommendations:Nomenclature in evaluation of analyticalmethods including detection and quan-tification capabilities (IUPAC recom-mendations 1995) as follows:36

Sensitivity = (No. true-positive samplesX 100) divided by (No. true-positivesamples + No. false-negative samples).

Specificity = (No. true-negative samplesX 100) divided by (No. true-negativesamples + No. false-positive samples).

Sensitivity and specificity of the newimmunosensor device calculated using

the above equations for results are100% and 100%, respectively.

CONCLUSIONBiosensor techniques are dynamic toolsfor determining different analytes forenvironmental, clinical, agricultural,food, veterinary, or military applications.Finally, a biosensor must be able to pro-vide a low and high detection limit witha rapid analysis time at a relatively lowcost.

The potential of the newimmunosensor device with a recombi-nant nucleocapsid antigen and a highlydispersed flow immunoelectrode for fastdetermination of antibodies againstSNV in mice blood has been shown. Thisapproach combined the advantages ofusing highly dispersed immunosorbent,highly sensitive electrochemical determi-nation of enzyme label, and flow-injec-tion scheme of immunoassay. A mainbenefit of recombinant nucleocapsidantigen application in immunoassay isthat antigen preparation is easy to stan-dardize. Since the principle of disposablesensing elements is involved, no regener-ation of immunosorbent between meas-urements is required. The immunosensordevice demonstrated a total assay timeof 22 minutes. This short overall assaytime could be a major advantage in fielddiagnostic screening of infectious dis-eases in mobile laboratories and smallhospitals. The electrochemical immuno-sensor may be used as a diagnostic toolfor screening large numbers of rodentserum samples for antibodies, diagnosisin humans, and distinguishing amonghantavirus serotypes. It is thought thatthis immunosensor device could poten-tially increase the sensitivity of detectionof antibody responses in the early stagesof hantavirus infection.

The top right-hand quadrant ofFigure 8 illustrated the positive bloodsamples. The bottom left-hand quadrantshowed negative blood samples. The


other 2 empty quadrants illustrated thatthere were no false-positive or false-neg-ative blood samples results when thestandard cyclic voltammetry or theimmunosensor device were used.

The results shown in Figure 5 reflecta good range from 1:400 to 1:500 dilu-tions. The 1:500 dilution was used in pre-vious work,22 and the authorsrecommend using the same dilution forfuture samples. However, the dilutionratio 1:200 showed similar strong differ-ences between strong-positive, weak-positive, and negative samples using thesame immunoreagent. Therefore a 1:200-1:500 dilution range was preferable.

The information about the back-ground current drift during measure-ments stage was programmed forautomatic subtraction of the steady-statesignal of the sample from its steady-statebackground current and was used as cor-rectional factor, as illustrated in Figure 5.

Other benefits of this type of assayinclude a disposable sensing element,very little use of reagents, and anincreased signal relative to theimmunosorbent assay, and the possibilityfor easy adaptation to detect otherviruses and bacteria.

Most of the existing limitationscould be directly related to operationaland/or long-term stability of the sensor’scomponents or the transducer itself.Other limitations could be attributed toreproducibility in complex matrices. Forpractical applications, the most impor-tant use of the sensors was encounteredonce the sensor was used for in situ realsample monitoring. An array of sensorsshould follow using microfluidics andnanotechnology.

ACKNOWLEDGMENTSThis work was supported by NSF grantDBI-0215384.

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