Impedance Sensor Arrays
for Development of a Real Time and Label Free Bio-Affinity Assay
Vena N. Haynes, Andrei L. Ghindilis, Mariya Smit and Holly M. Simon
Introduction
Several molecular biology techniques, including DNA microarrays, are used to detect and
characterize bacterial species in environmental samples based on nucleic acid hybridization.
Microarrays are useful for throughput analyses when comparing environmental factors and
describing the function of bacterial populations. However, microarrays have some issues
including non-specific binding of targets to probes and quantification of fluorescence label readouts (Wagner et al., 2007). Steric hindrances may also limit the binding of specific targets, and
excessive nucleic acid concentrations can disrupt the sensitivity of the detection (Zhou and
Thompson, 2002). At least some of these issues may be solved by using a simple low-cost
approach for the detection of nucleic acid hybridization with an impedance biosensor array.
Impedance sensor arrays do not require an expensive fluorescent label, and are able to rapidly
produce real-time data. Real-time detection provides information on the DNA binding and
hybridization kinetics.
The development of an impedance sensor requires the mass-production of the sensor
platform as well as a specialized reader, which in this case were both designed by Sharp
Laboratories of America (SLA). The current study shows the successful testing of the new
version SLA platform with a new DNA probe functionalization technique, achieved by
optimizing different parameters of electrochemical reading and assay conditions. We also set out
to show the applicability of these sensors to be used for genomic assays with double-stranded
DNA targets of three model genes. The genes were selected for their detection of various
Escherichia coli strains, including pathogenic ones. The first gene, adenylate kinase (adk) is a
housekeeping gene that is able to detect the presence of all E. coli strains. The other two genes,
hemolysin A (hlyA) and shiga toxin 2 (stx2) are virulence genes, chosen to detect specific
enteropathogenic (EPEC) E. coli strains. The future goal for these impedance biosensors is the
potential to quickly analyze environmental samples that contain a variety of microbial
organisms.
Materials and Methods
Preparation of DNA targets
E. coli strain CFT073 was used for genes adk and hlyA with an annealing temperature of
60oC and E. coli strain EDL933 was used for the gene stx2 with an annealing temperature of
50oC for PCR amplification. Both forward and reverse primers were used to produce doublestranded targets that are complimentary to the following sequences:
adk: 5’-AGTTCATCATGGAGAAATATGGTATTCCGCAAATCT-3’
hlyA: 5’-TGACTATTATGAAGAAGGAAAACGTCTGGAGAAAA-3’
stx2: 5’-CGGATTGCGCTAAAGGTAAAATTGAGTTTTCCAAG-3’
Double-stranded PCR product was purified using Promega Wizard Kit (Promega Corporation,
Madison, WI).
Optimizing sensors
Sensors were run on the SLA v3.0 Reader platform and were functionalized with adk
probes. During these experiments, chambers 1 and 3 were injected with a non-specific dsDNA
target, eluted with buffer (SSPE), and then injected with the specific adk dsDNA target.
Chamber 2 was injected with specific adk dsDNA target and then eluted with buffer. dsDNA
targets were denatured by heating at 95oC for 5 minutes prior to injection. Real-time impedance
data was analyzed in Arendar.
Results
Parameter:
Target concentration
Buffer (SSPE) concentration
Voltage (excitation potential)
Temperature
Tested:
2.5 µg/ml and 0.5 µg/ml
1X, 2X and 4X
40, 75, 100 and 150 mV
47oC and 52oC
Selection:
0.5 µg/ml
2X
75 mV
52oC
Table 1: Results from testing different parameters on adk-functionalized sensors for optimizing
conditions.
Injection
Figure 1: Impedance (Ω) versus time (sec) of the adk-functionalized sensors after parallel
injection of dsDNA targets. Injection of dsDNA targets was at 1115 seconds. Red channels
represent injection of the specific adk target, blue channels represent injection of the non-specific
hlyA target, and green channels represent injection of the non-specific stx2 target. Injection of
the specific adk dsDNA targets showed a higher rate of the impedance increase post-injection in
comparison to the non-specific targets and buffer baseline.
Injection of
buffer
Injection of
stx2
Injection of
Stx2
Injection of adk
Figure 2: Impedance (Ω) versus time (sec) of the adk-functionalized sensor during a sequential
injection of dsDNA targets. Sequential injections into the same chamber occurred as follows: (i)
a non-specific target, (ii) buffer, and then (iii) the specific target. Injection of the non-specific
(stx2) target showed no change, and a negative impedance response in comparison to buffer
baseline. In contrast, the impedance increased post-injection of the specific adk target around
2600 seconds, showing both the rate and intensity of binding between DNA targets and probes.
* Figures show sensors that were run at 75 Hz, 75 mV, 52oC with 2X SSPE buffer and 0.5 µg/ml
target concentration.
Discussion
In order to optimize sensors and assay conditions, data was analyzed based on an
assessment of response selectivity, comparing specific versus non-specific targets. The tested
parameters were chosen based on the maximum specific response and the minimum non-specific
response. The target concentration is important when considering the selectivity of binding
between the DNA probe and target. The concentration 0.5 µg/ml was optimum for the high level
of specific hybridization and low non-specific binding and was selected because the negative
control still gave reliable signal discrimination while producing a high specific response. It was
the lower one of two concentrations tested and provided for a better performance than the higher
concentration due to the lack of the steric hindrance caused by sensor saturation (Ghindilis et al.,
2009). The lower concentration was also chosen because it requires fewer targets and therefore
faster target preparation. Another assay parameter, the buffer concentration, did not affect the
signal response, and so a mid-range concentration was chosen. The different voltages that were
tested showed variable specific binding responses, and 75 mV statistically generated the best
impedance results with the least amount of background drift. Lastly, the 52oC temperature was
chosen as an assay condition because it is just below the melting temperature of the specific
DNA probe-target hybrid, thereby providing good assay selectivity. The temperature parameter
should be optimized for each assay separately because it depends on the probe sequence (the
thermodynamic parameter), and because it directly affects the hybridization reaction (Brewood et
al., 2008). Choosing a temperature close to the melting temperature of the specific target-probe
hybrid increases the dissociation of the mismatched DNA hybrids.
The ability of the sensors to be used for genomic assays was facilitated by optimization
of the assay conditions. The adk-functionalized sensors show high impedance upon injection of
complementary DNA targets in comparison to non-complementary targets (Fig. 1). Because the
mathematical algorithm used to quantify results is not yet fully available, comparative analysis of
this data is subjective. However, visually the data shows a steeper impedance slope for the
specific target, suggesting an increased hybridization rate. The non-complementary targets show
impedance lower than the buffer baseline, and sequential injections into the same chamber
demonstrate the difference in impedance between specific and non-specific targets (Fig. 2). For
all tests the negative control, stx2, had negligible signal. HlyA did show relatively high nonspecific binding and was a major challenge in assay optimization (data not shown).
The next step in this project is to confirm that sensors functionalized with hlyA and stx2
probes are just as selective and produce similar impedance results as adk-functionalized sensors.
This is important in particular because hlyA and stx2 are virulence genes, and the future goal for
these sensors is to be used on environmental samples to detect pathogenic bacteria species. Stx2
as an example is present in genomes of the infecting strains O157 that have been shown to cause
severe disease in human populations (Beutin et al., 2004). Development of an assay to quickly
analyze water samples could reduce risks associated with pathogenic bacteria. As impedance
sensors are optimized further, they will be used to rapidly characterize an array of bacteria
contained in a variety of water samples.
Literature Cited
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