Oral Fluid Nanosensor Test (OFNASET)

with Advanced Electrochemical-Based

Molecular Analysis Platform
VINCENT GAUa AND DAVID WONGb
a GeneFluidics Inc., Monterey Park, California, USA
b Division
of Oral Biology and Medicine, School of Dentistry at UCLA,
Los Angeles, California, USA

ABSTRACT: High-impact diseases, including cancer, cardiovascular dis-

ease, and neurological disease, are challenging to diagnose without sup-
plementing clinical evaluation with laboratory testing. Even with labo-
ratory tools, definitive diagnosis often remains elusive. The lack of three
crucial elements presents a road block to achieving the potential of clin-
ical diagnostic tests: (1) definitive disease-associated protein and genetic
markers, (2) easy and inexpensive sampling methods with minimal dis-
comfort for the subject, and (3) an accurate and quantitative diagnostic
platform. Our aim is to develop and validate a solution for requirement
(3) and also to develop a portable system. Requirements (1) and (2) will
be addressed through the utilization of novel and highly specific oral
cancer saliva proteomic and genomic biomarkers and the use of saliva
as the biofluid of choice, respectively. The Oral Fluid NanoSensor Test
(OFNASET) technology platform combines cutting-edge technologies,
such as self-assembled monolayers (SAM), bionanotechnology, cyclic en-
zymatic amplification, and microfluidics, with several well-established
techniques including microinjection molding, hybridization-based de-
tection, and molecular purification. The intended use of the OFNASET
is for the point of care multiplex detection of salivary biomarkers for
oral cancer. We have demonstrated that the combination of two salivary
proteomic biomarkers (thioredoxin and IL-8) and four salivary mRNA
biomarkers (SAT, ODZ, IL-8, and IL-1b) can detect oral cancer with
high specificity and sensitivity. Our preliminary studies have shown com-
pelling results. We sequentially delivered a serial dilution of IL-8 antigen,
probe solution, wash, enzyme solution, wash, and mediator solution to
sensor reaction chambers housed in a prototype cartridge and demon-
strated strong signal separation at 50 pg/mL above a negative control.

Address for correspondence: Vincent J. Gau, GeneFluidics, Inc., 2540 Corporate Place, Suite B101,
Monterey Park, CA 91754. Voice: 323-269-0900; fax: 323-269-0988.
[email protected]

Ann. N.Y. Acad. Sci. 1098: 401–410 (2007). 

C 2007 New York Academy of Sciences.

doi: 10.1196/annals.1384.005
401
402 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

KEYWORDS: salivary diagnostics; electrochemical detection; cyclic en-

zymatic reaction; bionanotechnology; genetic assay; immunoassay; si-
multaneous multichannel detection; point-of-care; PCR-less detection;
ultrasensitive detection

INTRODUCTION

Microfabrication technology has enabled the development of electrochem-

ical biosensors with the capacity for sensitive and marker-specific detection
of nucleic acids and proteins.1–5 The ability of electrochemical sensors to di-
rectly identify nucleic acids or proteins in complex mixtures is a significant
advantage over approaches, such as PCR, that require target purification and
amplification. Application of universal molecular analysis to cancer screening
has the potential for recognition of cancer-specific signature sequences and
protein markers in biological fluids. The most challenging body fluid submit-
ted to clinical microbiology laboratories is saliva on account of its complexity
and the low concentration of analytes. PCR-based methods for purification and
identification of cancer-related genetic targets are time-consuming and labor-
intensive. Oral cancer is the sixth most common cancer in the United States,
affecting 38,000 Americans annually and killing 7,200. Worldwide, oral cancer
annually affects 350,000 individuals (http://www.oralcancerfoundation.org/).
Over 90% of these cancers are squamous cell carcinoma. Despite treatment ad-
vances that have resulted in reductions in patient morbidity, the overall 5-year
survival rate for oral squamous cell carcinoma (OSCC) remains among the
worst of all cancer death rates (approximately 30–40%), considerably lower
than survival rates for colorectal, cervical, and breast cancer.6,7
A rapid, automated, point-of-care system using microfluidic and micro-
electro-mechanical systems (MEMS) technologies for measuring DNA, gene
transcripts (mRNA), proteins (cardiac and periodontal disease markers), elec-
trolytes, and small molecules in saliva for cardiovascular disease would have
a significant impact on the future development of salivary diagnostics.
A general approach for cancer-specific identification of genetic material
(DNA, RNA) or protein markers using an electrochemical sensor involves hy-
bridization of single-stranded oligonucleotide capture and detector probes to
target 16S rRNA2,8 or affinity binding of antibodies to cancer-related anti-
gens. The capture probe anchors the target to the sensor, while the detector
probe signals the presence of the target through a reporter molecule (FIG. 1).
Both oligonucleotide probes will be replaced by antibody pairs for protein
marker detection, such as thioredoxin or IL-8, as shown in FIGURE 2. Binding
of the capture and detector probes to the nucleic acid target creates a three-
component “sandwich” complex on the sensor surface.9–12 The fluorescein-
modified detector probe or antibody enables binding of an antifluorescein-
conjugated horseradish peroxidase (HRP) reporter enzyme to the target–probe
GAU & WONG 403

FIGURE 1. The detection of a salivary genetic marker after lysing the cells by mixing
saliva with lysis buffer.

complex.8 Addition of a redox substrate and application of a fixed potential

between the working and reference sensor electrodes creates a HRP-mediated
redox cycle that is detected by the electrochemical sensor as current.13,14 In this
way, the amplitude of the electroreduction current reflects the concentration
of the target–probe complexes on the sensor surface. Both genetic and im-
munoassays share the same reagents except for the binding solutions (capture
and detector), and it is possible to multiplex different assays onto the sensor
array chip for simultaneous multianalyte detection.
Initial proof-of-concept result has indicated that this electrochemical sensor
approach has the potential for detection of cancer-specific protein markers in
saliva while preliminary clinical studies have identified markers for infectious
disease in urine.15,16

MATERIALS AND METHODS

Sensor Preparation for IL-8 Immunoassay

Microfabricated electrochemical sensor arrays with an alkanethiolate self-

assembled monolayer (SAM) were obtained from GeneFluidics (Monterey
Park, CA, USA). SAM integrity was confirmed by cyclic voltammetry (CV)17
using a 16-channel potentiostat (GeneFluidics). After CV characterization,
404 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 2. The detection of a salivary proteomic marker, such as thioredoxin of IL-8

directly from saliva without pretreatment.

sensor arrays were washed and dried. Washing steps were carried out by ap-
plying a stream of deionized H 2 O to the sensor surface for approximately 2–3
sec followed by 5 sec of drying under a stream of nitrogen. To functionalize
the sensor surface, 4 L of 1.04 mg/mL polyclonal human anti-IL-8 capture
antibodies in 1× phosphate-buffered saline (PBS) with 0.1% sodium azide was
added to the alkanethiol-activated sensors. After 30 min of incubation at room
temperature, the sensor array was washed and dried, completing the surface
preparation.

Amperometric Detection of IL-8 Protein Marker

For each individual sensor of a 16-sensor array chip, 4 L of the biotinylated

IL-8 antibody (12.5 g/mL) in 1× PBS, was mixed with the same volume of
saliva. The antibody–saliva lysate mixture was applied onto each sensor and
incubated for 30 min to allow affinity binding of antibody to IL-8 analyte in
saliva. After washing and drying, 4 L of streptavidin-conjugated POD 1.25
g/mL or 13.4 nM; Zymed, CA, USA) in 0.5% BSA in 1× PBS was deposited
on each of the working electrodes for 15 min. After washing and drying, a
prefabricated plastic well manifold (GeneFluidics) was bonded to the sensor
array. Eighty microliters of HRP substrate solution (K-Blue Aqueous TMB;
Neogen, Lexington, KY, USA) was placed on each of the sensors in the array
so as to cover all three of the electrodes. Measurements were immediately
and simultaneously taken for all 16 sensors. The entire assay protocol was
GAU & WONG 405

completed within 45 min from the initiation of saliva mixing. Amperometric

current versus time was measured using a multichannel potentiostat (Gene-
Fluidics). The voltage was fixed at –200 mV (vs. reference), and the electrore-
duction current was measured at 60 sec after the HRP redox reaction reached
steady state.

Sensor Preparation for IL-8 RNA Genetic Assay

The integrity of the SAM chip was confirmed by CV using a 16-channel

potentiostat (GeneFluidics). To functionalize the sensor surface, 4 L of
0.5 mg/mL streptavidin (Calbiochem, San Diego, CA, USA) in H 2 O was added
to the alkanethiol-activated sensors, incubated for 10 min at room temperature
and washed. Biotinylated capture probes (4 L, 1 M in 1 M phosphate buffer,
pH 7.4) were added to the streptavidin-coated sensors. Phosphate buffer (1 M),
pH 7.4, was prepared by mixing 1 M NaH 2 PO 4 and 1 M K 2 HPO 4 in a 19:81
(vol/vol) ratio, respectively, and adjusting the pH to 7.4. After 30 min of incu-
bation at room temperature, the sensor array was washed and dried, completing
the surface preparation.

Amperometric Detection of IL-8 RNA Molecule

Salivary RNA samples were obtained from Dr. David Wong’s laboratory
at the University of California, Los Angeles. Fifty microliters of the detec-
tor probe (0.25 M) in 2.5% bovine serum albumin (Sigma)–1 M phos-
phate buffer, pH 7.4, was added to the salivary RNA sample. The detector
probe/sample mixture was incubated for 10 min at 65◦ C to allow hybridization
of the detector probe to target rRNA. Four microliters of the sample/detector
probe mixture was deposited on each of the working electrodes in the sensor
array. The sensor array was incubated for 15 min at 65◦ C in a humidified
chamber. After washing and drying, 4 L of 0.5 U/mL anti-fluorescein HRP
Fab conjugate (Roche), diluted in 0.5% casein in 1 M phosphate buffer, pH 7.4,
was deposited on each of the working electrodes for 15 min. After washing and
drying, a prefabricated plastic well manifold (GeneFluidics) was bonded to the
sensor array. Eighty microliters of HRP substrate solution (K-Blue Aqueous
TMB) was placed on each of the sensors in the array so as to cover all three
of the electrodes. Measurements were immediately and simultaneously taken
for all 16 sensors. Amperometric current versus time was measured using a
multichannel potentiostat (GeneFluidics). The voltage was fixed at –200 mV
(vs. reference), and the electroreduction current was measured at 60 sec after
the HRP redox reaction reached steady state.
406 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 3. Six-channel immunoassay schematic of RSC and FCC.

The Oral Fluid NanoSensor Test (OFNASET) Cartridge

The microfluidics cartridge was designed to enable all steps in a manual

assay protocol to be conducted in a “hands free” format. When the cartridge
is loaded with reagents specific for a panel of assays, such as those for oral
or breast cancer detection, the cartridge can serve as a disposable device for
saliva-based diagnostics.
The current prototype has a microfludic cartridge with two main parts-–
a top piece or reagent storage cartridge (RSC), which would house all of
the reagent and sample reservoirs, and a bottom piece or fluidic channel
cartridge (FCC), which would house all of the fluidic channels, reaction cham-
bers, and valve structures. FIGURE 3 shows an interface schematic of the two
pieces and FIGURE 4 shows an FCC prototype cartridge with RSC unit in the
back.
While still in development, RSC and FCC can be integrated to form the
OFNASET–Cartridge to be inserted into the reader Instrument and initiate
the experiment through an instrument interface where actuators depress the
flexible sample, reservoir. Simultaneously, the sample passive valve (described
below) would be opened for each of the sensor chambers. The resulting pressure
would drive the sample from the RSC to the FCC through the pin bump structure
and onward to each sensor chamber with an open valve. Note that without the
opening of the valves, the sample would remain in the sample reservoir. Note
further that air in the microfluidic channels would be vented through venting
channels.
GAU & WONG 407

FIGURE 4. Prototype cartridge of FCC and RSC.

For protein or cell-surface antigen detection, the detecting antibody solution

would be introduced to each sensor chamber and mixed with the sample. The
capture antibodies would immobilize the target–signal antibody complexes
onto the sensor surface before the wash solution was delivered to remove non-
bound material. A sensor chamber “ramp structure” would enable a consistent
back-pressure to make the sheer force wash more efficient. Enzyme solution
would then be delivered to each chamber, which would bind to the signal anti-
bodies. A second wash would remove nonbound enzyme. During the last step,
the substrate solution would be delivered and the bias potential applied through
the electrode interface. Amperometric current would be measured at 60 sec.
A postassay CV would provide a baseline signal for calibration of the specific
sample, sensor, and reagents. All waste solutions would be routed back to the
RSC.

RESULTS

IL-8 Protein Detection in Saliva Using FCC

To date, most of our assays have been conducted with the sensor chip and
reader (manual sample preparation). We have also developed the use of FCC
for automated sample preparation. These studies have shown preliminary but
compelling results. In one example, we have demonstrated the successful se-
quentially delivered serial dilution of IL-8 antigen, probe solution, wash, en-
zyme solution, wash, and mediator solution to sensor reaction chambers housed
in a prototype cartridge. As shown in FIGURE 5, the experiment demonstrates
strong signal separation at 50 pg/mL above a negative control. Note the smaller
error bars due to the minimization of error from manual handling.
408 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 5. Detection of IL-8 protein using FCC.

Use of the OFNASET Sensor Chip for Salivary IL-8 Protein

and RNA Detection

Saliva samples from patients with oral cancer and matched controls were
tested with GeneFluidics’s electrochemical-based detection platform; IL-8 pro-
tein and IL-8 RNA were the targets. The results shown in FIGURE 6A to C
demonstrate a clear separation between the signal from the cancer patients and
the controls. Each sample was tested in duplicate and no pretreatment was
conducted on the saliva prior to testing, although RNA was isolated prior to
detection.

DISCUSSION

We have developed a sensor array chip for direct electrochemical detection

of the cancer markers (RNA and protein) from saliva associated with oral
cancer. The sensor assay system relies on efficient binding of target RNA
molecules or proteins onto the sensor surface. This sensor array chip is part of
the developmental work for production of the final microfluidics cartridge OF-
SANET. The detection system involves oligonucleotide capture and detector
probes or antibodies that bind to RNA or antigens present in saliva. Prelim-
inary results show validation of rapid salivary diagnostics for point-of-care
applications. The initial study was conducted using IL-8 RNA and protein as
the cancer marker targets. Additional cancer markers, such as thioredoxin-
1 (protein), OAZ1 (RNA), SAT (RNA), S100P (RNA), DUSP1 (RNA), and
HIST3H3 (RNA), will be tested and validated with clinical specimens to further
expand the oral cancer test panel using this electrochemical-based detection
platform.
GAU & WONG 409

ACKNOWLEDGMENTS

We thank other members of the UCLA OFNASET team—David Wong,

Chih-Ming Ho, and Jim Dow at Becton Dickson—for joint cooperation. We
also thank David Haake and Bernard Churchill for helpful discussions and
long-term collaboration on assay development. The development of this study
was supported by U01 Grant DE17790 from the National Institute of Dental
and Craniofacial Research (NIDCR) and Bioengineering Research Partner-
ship Grant EB00127 (to B.M.C.) from the National Institute of Biomedical
Imaging and Bioengineering. The primary author is the founder and majority
shareholder of GeneFluidics.

REFERENCES

1. DRUMMOND, T.G., M.G. HILL & J.K. BARTON. 2003. Electrochemical DNA sensors.
Nat. Biotechnol. 21: 1192–1199.
2. GAU, J.J., E.H. LAN, B. DUNN, et al. 2001. A MEMS based amperometric detector
for E. coli bacteria using self-assembled monolayers. Biosens. Bioelectron. 16:
745–755.
410 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

3. GOODING, J.J. 2002. Electrochemical DNA hybridization biosensor. Electroanaly-

sis 14: 1149–1156.
4. PALECEK, E. & F. JELEN. 2002. Electrochemistry of nucleic acids and development
of DNA sensors. Crit. Rev. Anal. Chem. 3: 261–270.
5. WANG, J. 2002. Electrochemical nucleic acid biosensors. Anal. Chim. Acta 469:
63–71.
6. SCHANTZ, S.P. 1993. Carcinogenesis, markers, staging, and prognosis of head and
neck cancer. Curr. Opin. Oncol. 5: 483–490.
7. SCHANTZ, S.P. 1993. Biologic markers, cellular differentiation, and metastatic head
and neck cancer. Eur. Arch. Otorhinolaryngol. 250: 424–428.
8. GAU, V., S.C. MA, H. WANG, et al. 2005. Electrochemical molecular analysis
without nucleic acid amplification. Methods 37: 73–83.
9. CAMPBELL, C.N., D. GAL, N. CRISTLER, et al. 2002. Enzyme-amplified ampero-
metric sandwich test for RNA and DNA. Anal. Chem. 74: 158–162.
10. DEQUAIRE, M. & A. HELLER. 2002. Screen printing of nucleic acid detecting carbon
electrodes. Anal. Chem. 74: 4370–4377.
11. UMEK, R.M., S.W. LIN, J. VIELMETTER, et al. 2001. Electronic detection of nucleic
acids: a versatile platform for molecular diagnostics. J. Mol. Diagn. 3: 74–84.
12. WILLIAMS, E., M.I. PIVIDORI, A. MERKOCI, et al. 2003. Rapid electrochemical
genosensor assay using a streptavidin carbon-polymer biocomposite electrode.
Biosens. Bioelectron. 19: 165–175.
13. FANJUL-BOLADO, P., M.B. GONZALEZ-GARCIA & A. COSTA-GARCIA. 2005. Am-
perometric detection in TMB/HRP-based assays. Anal. Bioanal. Chem. 382:
297–302.
14. MECHERI, B., L. PIRAS, L. CIOTTI & G. CAMINATI. 2004. Electrode coating with
ultrathin films containing electroactive molecules for biosensor applications.
IEEE Sens. J. 4: 171–179.
15. SUN, C.P., J.C. LIAO, Y.H. ZHANG, et al. 2005. Rapid, species-specific detection of
uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridiza-
tion and an electrochemical sensor array. Mol. Genet. Metab. 84: 90–99.
16. LIAO, J.C., M. MASTALI, V. GAU, et al. 2006. Use of electrochemical DNA biosen-
sors for rapid molecular identification of uropathogens in clinical urine speci-
mens. J. Clin. Microbiol. 44: 561–570.
17. BARD, A.J. & L.R. FAULKNER. 2001. Potential Sweep Methods.: 226–260. John
Wiley & Sons. Hoboken, New Jersey.