Andrey Ghindilis

Real-Time Biosensor Platform: Fully Integrated Device for Impedimetric Assays

An impedimetric biosensor platform for bioaffinity assays has been developed that is based on real-time, label-free electrochemical detection performed via a direct interface to electronic digital data processing. The sensor array consists of 15 gold microelectrode pairs (Fig. 1) that are enclosed in three reaction chambers and biofunctionalized with specific probes. The impedance change caused by specific capture of target analyte molecules on the functionalized electrode surface is recorded in real time. The measuring instrument is capable of continuous and simultaneous stimulation and recording of all electrodes on the array. A corresponding mathematical algorithm and a software package for data analysis have been developed. The software performs (i) filtering of the instrument noise, and (ii) extraction of the exponential component of the impedance signal. Thus, the algorithm can quantify both rate of target to probe binding, and target to probe affinity. The described fully integrated platform can be used as a basic research tool for development of various bio-affinity impedimetric assays. To facilitate such applications, we have developed a streamlined manufacturing technology, and a set of assay protocols for detection of microbes based on nucleic acid hybridization. The assay was shown to detect and distinguish between two closely related but different Escherichia coli strains. The assay sensitivity was sufficient for reliable measurements of specific PCR products amplified from microbial genomic DNA. The sensor array platform is adaptable for detection of a wide range of analytes of practical significance, and it has potential for further integration with amplification (i.e. PCR) and sample preparation modules.

Electrochemical Detection on Microarrays

Microarray detection methods have long been based upon optical methods: visible detection, fluorescence, luminescence, and surface plasmon resonance (1–6). In the case of the visible detection method an enzyme produces a substrate that forms an insoluble precipitate at the spot site, which becomes visible to the naked eye. Surface plasmon resonance also uses visible light, but in this case the energy (and reflected angle) of the light absorption on the metal surface is altered; the researcher finds the absorption minimum using an array detector. This method utilizes more expensive equipment in the process of detecting the material on the chip/slide (4). More sensitive methods such as fluorescence and luminescence have also been employed for the detection of material on the chip/slide surface. The fluorescencebased system requires a laser, so the beam can be rastered over the chip area. Or conversely, the chip can be illuminated with light and then a signal detected with a CCD camera using the appropriate filters (3). Similarly, luminescence detection utilizes a CCD camera after an enzyme (such as alkaline phosphatase) has generated a product that luminesces (1). Both these methods require expensive optics and equipment that may cost

Impedimetric biosignal analysis and quantification in a real-time biosensor system

60,000 to

Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR

500,000. Moreover, the footprint of the larger and more expensive systems requires a benchtop area on one side of the room. A less expensive, mobile, and more sensitive platform for microarray detection purposes is highly desirable. To this endeavor, many researchers in the field have instituted programs for electrochemical detection on microarrays (6–20). In order to do this, one must have active electrodes on the chip or glass surface. Thus many of the spotted slides out in the marketplace contain surface areas where the spots are not electroactive and do not qualify for electrochemical detection.

An improved algorithm for quantifying real-time impedance biosensor signals

We describe our real-time, label-free, electrochemical impedance biosensor system with an emphasis on the use of an impedance response signal model to quantify assays. The signal processing for estimating model parameters from noisy data and the quantitative verification against target concentration and affinity are also presented.

Use of Redox Enzymes for the Electrochemical Detection of Sequence-Specific DNA and Immunochemical Entities

An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10−17 M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

In previously published work [1] we presented a real-time electrochemical impedance biosensor prototype system and a state-space estimation algorithm for signal quantification. Experiments in the interim have revealed some algorithm failure modes which reduced the reliability and repeatability of quantification. The present work describes a related algorithm that introduces constraints based on a priori knowledge of the expected signals predicted by the biosensor signal model. The improvements in reliability and repeatability bring the system close to deployment for real-world trials.

Immunoassays based on electrochemical detection using microelectrode arrays.

There is a host of methods for the detection of immunoassays and sequence-specific DNA on a microarray chip (or biochip); see earlier chapter. Many detection technologies are optical methods, such as surface plasmon resonance, luminescence, fluorescence, and visible detection modes (absorbance or reflectance). They do require optical systems that are somewhat expensive. As shown in previous chapters, electrochemical detection is a viable option for immunochemical and sequence-specific DNA detection. The electrochemical detection methods vary greatly, with anything from impedance measurements, to oxidation of specific nucleotdides within the duplex, to conductive interacalators, redox-intercalators, metal tags, and redox enzyme systems. Many have specific niches and amplification modes. The bottom line is that electrochemical detection is sensitive, the system footprint is small, and the system is inexpensive. To date only a few electrochemical detection systems have been commercialized and succeeded. These include (1) the CombiMatrix ElectraSense system, utilizing HRP as a redox enzyme (1–6); (2) the Osmeotech Esense (Motorola Life Sciences) system, utilizing a Ferrocene tag (see Chapter 12 in this book); and (3) the Toshiba gene analyzer, utilizing a redox active dye (7). The CombiMatrix system is the only commercial system that uses a true microarray concept, up to 12,000 individual electrodes. The latter E-chem detection systems utilize a microarray that requires spotting onto gold electrodes and the number of electrodes is in the neighborhood of 15–25 per chip. In these cases, the voltage is scanned and current recorded. A substantial increase in the peak-to-peak current reflects the presence of the redox species present, and hence a duplex being formed. In the case where redox enzymes (or products that are redox active) are utilized for the detection of specific DNA sequences and immunoassays, there is a variety of enzymes from which to choose. Many have been used before on simple electrode