15

Review
Electrochemical Biosensors for DNA Sequence Detection
Susan R. Mikkelsen*
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, PQ, Canada H3G 1M8

Received: April 16, 1995

Final version: May 2, 1995

Abstract
A review of reported biosensors for the sequence-selective detection of analyte DNA sequences shows that signal transduction based on
electrogenerated chemiluminescence, potentiometry and voltammetry are all capable of detecting analyte DNA at attomole levels. In
contrast, optical and piezoelectric transducers detect DNA at picomole and femtomole levels, respectively. The principles and applications
of these promising electrochemical DNA biosensors are described and discussed.

Keywords: Electrochemical biosensors, DNA sequence detection, Chemiluminescence, Potentiometry, Voltammetry, Attomole levels

1. Introduction
The detection of specific base sequences in human, viral and
bacterial nucleic acids is becoming increasingly important in the
diagnosis of disease [l-51. The mutations responsible for
numerous inherited human disorders are now known, and this
knowledge is steadily increasing as the sequencing of the human
genome continues. Pathogens responsible for disease states,
bacteria and viruses, are also detectable via their unique nucleic
acid sequences, and interest in their detection continues to grow.
Table 1 gives a representative listing of some applications of
sequence-selective DNA detection methods.
Human DNA, with a six-billion base-pair sequence present in
46 chromosonies, can be extracted from white blood cells, which
are present at levels of about 7-8 million cells per mL of blood
original analyte DNA strands define the length of the amplified
segment of DNA, and successful amplifications of 40- to 2000-
base fragments have been achieved.
When genomic DNA has been isolated and, if necessary,
amplified by PCR, it can be examined using a bioassay or a
biosensor that is selective for a particular base sequence. For the
purposes of this review, a DNA biosensor is considered to be a
device in which analyte DNA is captured at a transducer
surface, and measurement proceeds with immobilized analyte
DNA. Following a brief discussion of bioassays and biosensors
for nucleic acid detection [S-221, this review focuses on
sequence-selective DNA biosensors that employ electrochemical
methods for signal transduction [23-301.
Table I . Examples of DNA diagnostic applications ( 1 -5, 81
~ - ~ ~~~ 7 ~

[6]. Each cell contains one or two copies of the sequence Source of D N A / R N A Application
responsible for a particular trait, so that 12 (or 24) attomoles of
analyte DNA are available in a 1 mL blood sample. Clearly, any Human Paternity/forensic testing
test applied to native human DNA requires exquisite selectivity HLA complex
D-loop region (mitochondria1 DNA)
as well as a very low detection limit.
length polymorphisms (VNTR loci)

Oncogenes/tumor suppressor genes

2. Sample Preparation c-myc, c-myb, c-abl, c-sis, c-ras, G-protein,
jun, p53 and retinoblastoma genes

The concentration of a particular sequence region of DNA in Inherited disorders

a test sample can be amplified, or increased, using the Cystic fibrosis
H ypercholesterolemia
polymerase chain reaction [7], or PCR (Fig. 1). This reaction Sickle-cell anemia
is performed in the presence of the four deoxynucleotide Huntington's disease
triphosphate monomers, the test DNA, the enzyme DNA Duchenne muscular dystrophy
polymerase from the thermophilic bacterium T h e r m u s aquaticus p-Thalassemias
(Taq DNA polymerase), and two oligodeoxynucleotide primers. Adult polycystic kidney disease
The primers are chosen to bracket the region of interest in the Hemochromatosis
Hemophilia A/Von Willebrand disease
test DNA, at the 5'-ends of the region of interest on the sense
and antisense strands, respectively. By precisely controlling the Viral Cytomegalovirus (DNA)
temperature of the reaction mixture, the test DNA can be made Human papillomaviruses (DNA)
to denature into single-stranded DNA (93"C), anneal with the Rotavirus (RNA)
primers (37755"C), and undergo primer extension to create one Human immunodeficiency virus (DNA)
Human t-cell leukemia viruses (DNA)
copy of the sequence of interest (70°C). The complete tem-
perature cycle may take as little as 2 min, and each amplification Bacterial Mycobacterium tuberculosis (DNA)
cycle will produce one copy of the sequence of interest from the Mycoplasma pneumoniae (RNA)
test and copied DNA. The amplification factor is thus 2" - I , Gonorrhea (RNA or DNA)
where n is the number of temperature cycles used, typically 20- Chfamjdiu (DNA)
30. In principle, amplification factors of 10' to lo9 can be Escherichia coli (RNA)
Baccilus suhtilis (RNA)
achieved in this manner, although the values are somewhat Baccilus hurgdorferi (DNA)
lower in practice. The positions of the two primers on the

Electrounalysis 1996, 8, No. 1 0 VCH Verlagsgeselischuft mbH, 0-69469 Weinheim, 1996 1040-0397/96/0101-015 $ 5 .00+ .25/0
16

--
--
-- Denature dsDNA

Anneal Primers
37-55 "C
S . R. Mikkelsen

An alternative method [9] designed to detect point mutations

(the substitution of a single base by another) employs two
primers complementary to a contiguous -50-base sequence on
the same analyte DNA strand. A thermostable ligase enzyme
will join the ~ 2 5 - b a s eprimers only if a perfect match between
primer and analyte DNA sequences occurs at the junction.
Detection of ligated products, or unligated primers, is accom-
plished by electrophoresis.

v- 4. Biosensors for Sequence-Selective DNA Detection

Extend Primers

--
Interest in biosensors capable of rapidly and selectively
detecting native DNA sequences has steadily increased [ l l , 121.
Biosensors are devices that combine a biological recognition

--
--
Denature dsDNA
Fig. I . Polymerase chain reaction (PCR) cycle.
3. Bioassays for Sequence-Selective DNA Detection
Bioassays for DNA employ labelled, single-stranded oligo-
nucleotides, complementary to the analyte sequence, for
detection. These species, called DNA probes [8], hybridize
with gel- or membrane-immobilized analyte DNA, and the
presence of the detectable label following rinse steps indicates
the presence of the DNA probe's complementary base sequence
in the analyte DNA. DNA probes must be at least 16 bases long,
to selectively recognize a unique human DNA sequence, and
probe lengths of 18 bases to several kilobases have been used.
Labels include 32P in the 5'-terminal phosphate group,
fluorophores, and biotin attached to the terminus or to bases,
where detection follows incubation with enzyme or fluorophore
conjugates of avidin or streptavidin [9].
DNA testing methods based on PCR and similar reactions
can be made highly selective, but are time-consuming, since
PCR itself requires a minimum of one hour, and this is added to
the time needed for analyte DNA isolation, DNA probe
hybridization and detection steps. Methods that avoid a
subsequent detection step with DNA probes have used PCR
primers designed to be selective for the analyte DNA sequence
[8], so that the presence or absence of a PCR product provides
the test result.
agent, which confers selectivity, with a transducer, which
provides sensitivity and converts the recognition event into a
measurable electronic signal. To date, biosensors for the
detection of nucleic acid sequences have used single-stranded
nucleic acids for recognition of the complementary sequence,
and transduction has been accomplished with fiber optic
fluorimetry [ 13, 141, reflectometry [15], surface plasmon
resonance [16], piezoelectric resonators [ 17, 181, the quartz
crystal microbalance [19, 201, a surface acoustic wave detector
[21,22], an electrogenerated chemiluminescence cell [23], a light-
addressable potentiometric sensor [24, 251, and voltammetric
sensors [26-301. Table 2 shows a comparison of the reported
figures of merit for these biosensors. In addition to biosensors
that exploit the natural selectivity of single-stranded DNA for
the detection of complementary sequences, biosensors for
DNA-binding drugs [311 and divalent magnesium [32], based
on their binding to DNA immobilized on electrode surfaces,
have also been reported.
This review focuses on three types of sequence-selective DNA
biosensor, all of which employ electrochemical methods for
signal transduction [23-301. The detection principles and
reported applications will be described, and new directions for
future work will be discussed.
5. Transduction Based on Electrogenerated
Chemiluminescence
This device allows the selective hybridization reactions to
occur in homogeneous solution, where they occur faster than in
the mass-transport-limited situation where one of the probe
strands is immobilized [33]. Two DNA probes are used, and
both are labelled at the 5'-terminus, one with biotin, the capture

Table 2. Survey of reported DNA biosensors.

~

Trunsducer Immobilization Probe D N A length Target D N A length Assay time Detection limit [a]
method [base] [base] [min] mass molar

Potentiometric (pH) [24, 251 Biotin-streptavidin 20 114 75 30 am01

Voltammetric (carbon) [29, 301 Covalent 18 4000 10 (1.8 fmol)
Voltammetric (gold) [26] Thiol chemisorption 20 4200 60 (0.1 amol)
Electrogenerated chemiluminescence [23] Biotin-streptavidin 20 110 25 lOamol
1829 200 amol
Surface acoustic wave [21, 221 Adsorption 4000 4000 180 (1.5fmol)
Quartz crystal microbalance [19, 201 Thiol chemisorption 10 7249 60 (10 fmol)
Optical fiber (fluor) [13, 141 Covalent 16 16 3 (1 pmol)
Surface plasmon resonance [16] Adsorption 17 97 5 1 fmol

[a] Values in parentheses were calculated from reported detection limits, using a formula weight of 330 g/mol base.

Electroanalysis 1996, 8, No. 1

Electrochemical Biosensors for DNA Sequence Detection
Counler Counfer
Electrode Electrode
Reference
Electrode
I for a short (1 10-base) PCR product, and 200 amol for a longer
+ +
@ for shorter fragments. This may result from diffusion-limited
Fig. 2. Flow cell including DNA biosensor based on transduction by
electrogenerated chemiluminescence. Reproduced from [23].
reagent for subsequent immobilization, and the other with a
tris(2,2'-bipyridyl)ruthenium chelate. The probes are designed
to function as PCR primers for amplification of the human p-
globin gene, which is the target, or analyte DNA sequence. The
double-stranded PCR product has biotin attached to the 5'-end
of one strand and the ruthenium chelate bound to the 5'-end of
the complementary strand. Following PCR amplification, the
double-stranded product is captured onto streptavidin-coated
magnetic polystyrene beads [23].
The heart of the biosensor is an amperometric flow cell (Fig.
2), where a magnetic arm moves to a position directly behind the
working electrode to capture and retain the magnetic beads at
the electrode surface. Once retained in this manner, the beads
are examined for the presence of the ruthenium chelate, which
emits light (620 nm) when oxidized in the presence tripropyla-
mine. The reactions involved in the electrochemiluminescence
detection scheme are shown below [34], in Equations 1-5:
Ru(bpy)? -+ Ru(bpy)if + e- (1)
(CH3CH2CH2)3N-+ (CH3CH2CH2)3N"e- (2)
( C H ~ C H Z C H ~' +) ~+N
H+ + (CH3CH2CH2)2N '+- CHpCH2CH3 (3)
Ru(bpy):+ + (CH3CH2CH2)2N.+- CH-CH2CH3 +
Ru (bpy):+* + (CH3CH2CH2)2 N+=CHCH2 CH3 (4)
Ru(bpy):+* + Ru(bpy)? + hv (620nm) (5)
Oxidized tripropylamine (Eq. 2) is converted to an unstable,
highly reducing intermediate via deprotonation (Eq. 3), which
reacts with the oxidized ruthenium chelate to generate the
chemiluminescent form (Eq. 4). A voltage ramp is applied to the
working electrode, and the instrument detects a peak in light
emission intensity, which is integrated over a two-second
interval. Emitted light is detected by a photomultiplier tube
located directly opposite the surface of the working electrode.
It is surprising that the ruthenium chelate label on the bead-
bound, double-stranded DNA has access to the electrode
surface for oxidation (Eq. 1). The large signals seen for the
shortest (1 10 base-pair) fragment suggest a different mechanism,
since most of the ruthenium chelate, attached to the 4.5pm
17
polystyrene beads through a nearly 40 nm long DNA fragment,
would not have access to the electrode surface if the magnetic
beads were rigidly held. It is possible that the beads retain some
mobility when held at the working electrode surface, or a freely
diffusing buffer component is oxidized and acts as a mediator to
oxidize bead-bound Ru(bpy)i+ to initiate the reactions that
ultimately yield light emission.
This sensor allows the quantitation of PCR products with a
linear dependence of log(integrated light intensity) on the
logarithm of the quantity of PCR product (in a 50 pL sample)
over three orders of magnitude, with detection limits of 10 amol
(1 829-base) product. The improved detection limits with shorter
PCR products are attributed to a greater bead-binding efficiency
competitive binding of free (excess) biotinylated primer with
biotinylated PCR product for streptavidin sites, since short
PCR products will compete more effectively by rapid diffusion.
6. Transduction with the Light-Addressable
Potentiometric Sensor
Two DNA biosensors using potentiometric transduction have
been reported by this group: one is designed to quantitate total
DNA [25], while the second is designed for sequence-selective
detection of PCR products [24]. Both rely on the capture of
biotinylated species onto streptavidin-coated membranes, and
the detection of a local pH change produced at the membrane-
covered sensor surface by an enzyme label (urease).
A diagram of the light-addressable potentiometric sensor is
shown in Figure 3. It consists of a lightly doped (n-type) silicon
plate, covered with a surface layer of silicon nitride, an
insulator. The photoresponsive insulator layer is in contact
with electrolyte (the substrate solution), and its position is fixed
with respect to a light-emitting diode. When the LED is turned
on and ofl' with a 50% duty cycle, the sensor surface is
alternately illuminated, and an AC photocurrent is generated.
The magnitude of the current is related to the potential of the
electrolyte-insulator surface, which is controlled by the solution
pH. With urea hydrolysis producing a continuous change in pH,
results are expressed as a change in potential with time, with
magnitudes as large as 5mV/s, where a 1 mV/s signal change
corresponds to a rate of change of pH of about 1 pH unit per
min.
The total DNA sensor [25] allows binding between a
thermally-denatured DNA sample, biotin-labelled single-
stranded DNA binding protein, a monoclonal anti-DNA-
urease conjugate, and streptavidin to occur in homogeneous
solution. Streptavidin is a tetrameric protein, with four biotin-
binding sites per molecule. The multivalency of streptavidin for
biotin allows capture of the reaction product, (streptavidin):
(biotin-DNA-binding protein):DNA:(anti-DNA-Urease), onto
a biotinylated nitrocellulose membrane. An excess of all
reagents are added to the denatured DNA sample so that the
DNA is quantitatively bound by the proteins, and thus
quantitatively labelled with urease. Urease hydrolyses urea to
ammonium, bicarbonate and hydroxide ions, and its high
turnover number results in a measurable increase of pH with
time when excess urea is present in a dilute buffer.
The single-stranded DNA binding protein possesses a binding
site size of between 33 and 65 bases, while anti-DNA reacts with
a 20-base-long site on single-stranded DNA. Thus, a strand
length of at least 85 bases is necessary for DNA quantitation.
Electroanalysis 1996, 8, No. 1
18
Stick
Vanable Bias Voltage
Elenrode
Enzyme
Substrate
1 I piasiic Plunger
Sensor
Nitrocellulose Membrane
Fig. 3. DNA biosensor using the light-addressable potentiometric sensor
for signal transduction. Reproduced from [25].
The authors report that DNA fragments 603 to 118 bases long
are quantitated with decreasing efficiency, and that short DNA
fragments (25-72 bases) inhibit detection of longer DNA
strands. The authors do not state whether the inhibition
mechanism involves competition for the DNA-binding proteins
(a situation where increased reagent concentrations would
eradicate the problem) or competition of short streptavidin-
labelled DNA fragments (too short to bind to the anti-DNA-
urease conjugate) for biotinylated sites on the membrane. The
authors report that DNA 872 bases long is quantitatively
bound, and a detection limit of 2pg was reported for calf
thymus DNA [25].
More recently, the same group has reported a scheme for the
sequence-selective detection of PCR products with the light-
addressable potentiometric sensor [24], using two DNA probes,
one labelled with biotin and the other with fluorescein, instead
of the DNA-binding proteins used in the total DNA sensor. In
this work, analyte DNA (PCR product) is denatured and
incubated with the two DNA probes. The probes anneal to the
same target DNA strand in different locations, and then
streptavidin is added to the solution to capture the probe-
target hybrids onto a biotinylated nitrocellulose membrane
filter. The membranes are incubated with an anti-fluorescein-
urease conjugate to quantitatively label fluoresceinated DNA
probes with urease. Urease activity is measured in the presence
of excess urea by the same method used in the total DNA sensor.
It is interesting to note that this sensor for selective PCR
product quantitation yields the largest signals for the shortest
PCR products, in contrast to the total DNA sensor. Three fmol
of a 100-base target DNA fragment yielded a response of over
800,uV/s, while the same molar quantity of a 7.76 kilobase
Electronnalysis 1996, 8, No. I
S. R. Mikkelsen
fragment gave a response of only 200pV/s. This signal
dependence on target length is attributed to the steric hindrance
of binding reactions during the hybridization, capture or
enzyme conjugate steps. The authors report a detection limit
of 30 amol, for a 114-base PCR product, with negligible
interference from irrelevant DNA sequences up to 10 pg levels,
and suggest that improved detection limits could be achieved
through the use of multiple fluorescein labels per probe and
smaller target fragments.
7. Voltammetric Transduction
7.1. Measurement of Peak Currents
Our work in this area was first reported in 1992, with the
development of a new electrochemical method for detecting
DNA covalently bound to carbon electrode surfaces [36, 371.
Double-stranded DNA was detected voltammetrically, in the
presence of tris(2,2'-bipyridyl)cobalt(rrr), a complex which had
been shown to associate reversibly through electrostatic
interactions with the minor groove of double-helical DNA
[38]. Electrode-bound DNA shows no electroactivity over the
+ 1.0 to -0.9V (vs. SCE) potential range, and the cobalt
complex is reversibly electroactive (le) with a formal potential
of + 0.11 V (vs. Ag/AgCl). Thus, voltammetry at the modified
carbon electrode, using a dilute solution of the cobalt complex,
shows preconcentration of the complex at the electrode surface,
resulting in larger peak currents than expected for a diffusing
redox couple.
Immobilization chemistry was later improved by employing
N-hydroxysulfosuccinimide (NHS) with the water-soluble
carbodiimide reagent used to activate carboxylate groups on
the glassy carbon electrode surface [30]. Single-stranded DNA,
covalently bound to these groups through deoxyguanosine
residues, was readily distinguished from double-stranded DNA
formed upon hybridization, by voltammetric peak current
magnitudes for M solutions of Co(bpy)z+. Furthermore,
both the hybridization and detection steps employ noncovalent
binding, and the original conditions at the electrode surface
could be regenerated by simply rinsing the sensor with hot,
distilled water, so that only single-stranded DNA remained
bound to the sensor surface. A model sensor consisting of a 20-
mer of thymidylic acid, enzymatically elongated with deox-
yguanosine residues to allow immobilization, showed selective
hybridization and detection of the complementary polydeox-
yadenylic acid, poly(dA), with no signal increase seen after
exposure to an alternating copolymer of deoxyadenosine and
thymidine residues, poly(dAdT).
Extension of this work to a practical application employed
carbon paste electrodes modified by the inclusion of 5% stearic
acid, and examined the cystic fibrosis AF508 deletion sequence
associated with 70% of cystic fibrosis patients and carriers [29].
By performing hybridization reactions at 43"C, it was possible
to detect the disease sequence, with the three-base deletion, but
not the normal DNA sequence contained in synthetic oligo-
deoxynucleotides. The detection limit obtained for a 4000-base
fragment of DNA was 2.5 ng, or 1.8 frnol.
A recent report [26] of a similar voltammetric DNA sensor
shows that a different immobilization method and the use of a
different indicator species yield much lower detection limits.
Hashimoto et al. labelled a single-stranded, synthetic DNA
probe with a terminal thiol group, allowing chemisorption to a
gold electrode surface. Detection of immobilized DNA was
Electrochemical Biosensors for DNA Sequence Detection
accomplished by linear-sweep voltammetry, following a 5-min
exposure to a lOP4M solution of the redox-active DNA
intercalant Hoescht 33258, a bis(benzimide) dye, followed by a
5-min rinse with buffer. Clearly, the dye interacts strongly
enough with immobilized DNA that the rinse step does not
remove DNA-bound dye, so that much smaller signals are
observed prior to the hybridization step that forms double-
stranded DNA at the electrode surface. A detection limit of
g/mL is reported for a 4200-base target sequence.
7.2. Measurement of Peak Potentials
Prior to their report of a DNA biosensor based on peak
current measurements for Hoechst 33258 oxidation, Hashimoto
and co-workers studied the effect of the DNA modification of
graphite electrodes on the voltaminetric peak potentials of
organic, redox-active DNA intercalants. Their work shows that
intercalant peak potentials shift in the presence of adsorbed
DNA, and that peak potentials can be used to distinguish
between immobilized single- and double-stranded DNA [27,28].
Their initial article 1281 reports a small shift in the voltammo-
gram of acridine orange at electrodes modified with adsorbed
DNA. A later article [27]reports that of 27 candidates, including
acridine orange, daunomycin shows the greatest shift in
oxidation peak potential (34 mV) between adsorbed single-
and double-stranded DNA. Daunomycin anodic peak potential
measurements were used to detect a target DNA sequence, and
1 pg/mL solutions of target and interferant DNA yielded peak
shifts of + 18 mV and + 3 mV, respectively.
8. Future Directions
Based on the detection limits of the DNA biosensors reported
to date, it seems clear that electrochemical biosensors for
selective DNA sequence detection have a very promising future.
Efforts to detect sequences present in genomic DNA, without
PCR amplification, should be undertaken. Voltammetric
sensors have been shown to have low enough detection limits
to allow examination of DNA without PCR amplification.
However, the effect of the analyte DNA strand length on
hybridization rates and measured signals must be examined,
since genomic DNA is highly polymerized. The sequence-
selectivities of the DNA sensors reported to date have not been
examined in much detail, and conditions for the selective
detection of deletions (e.g., cystic fibrosis), point mutations (e.g.,
sickle-cell anemia), and extensions by repeat sequences (e.g.,
Huntington’s disease) must be established. Sensor arrays could
then be designed for rapid multilocus DNA screening, without
the need for separate PCR protocols for each analyte sequence.
It may also be possible to use an electrode-immobilized oligo-
deoxynucleotide as a PCR or ligase primer for rapid, in situ
detection of selective hybridization and primer extension/
ligation. Fundamental comparisons of hybridization kinetics
in homogeneous and heterogeneous reactions could also be
done with these devices.
19
9. Acknowledgement
Financial support from the Natural Sciences and Engineering
Research Council of Canada is gratefully acknowledged.
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Electroanalysis 1996, 8, No. 1