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1.3.1. Microarray Systems

Within the last couple of decades, the development of integrated biosensors for the detection of multiple biologically relevant species has begun to take place. These integrated biosensor arrays that use the same excitation source for all of the elements and the same measurement process have been termed many things; gene chips, DNA-chips, etc. Most of the different array chips have been based on the use of nucleic acids (i.e. DNA) as the bioreceptors. Figure 1.4 illustrates an example of DNA microarray system with its associated detection system. Other types of bioreceptors such as antibodies, enzymes and cellular components can also be used. It is noteworthy that substrates having microarrays of bioreceptors are often referred to as biochips although most of these systems do not have integrated microsensor detection systems. A few of the more recent applications and advances in biochip technology will be discussed in this review.

A microarray of electrochemical biosensors has been developed for the detection of glucose and lactate on line [54]. This array of electrochemical biosensors was prepared using photolithographic techniques, using glucose oxidase and lactate oxidase as the biore-ceptors. The glucose oxidase or lactate oxidase at each of the different sites in the array produces hydrogen peroxide when its appropriate substrate, glucose or lactate, is present. The hydrogen peroxide produced was measured at each element amperometrically.

An optical microarray system using a charge-coupled device (CCD) detector and DNA probes has been developed by Vo-Dinh and coworkers [48]. The evaluation of various system components developed for the DNA multi-array biosensor was discussed. The DNA probes labeled with visible and near infrared (NIR) dyes are evaluated. Examples of application of gene probes in DNA hybridization experiments and in biomedical diagnosis (detection of the p53 cancer suppressor gene) illustrated the usefulness and potential of the DNA

FIGURE 1.4. Schematic diagram of a DNA microarray with detection system.

FIGURE 1.4. Schematic diagram of a DNA microarray with detection system.

multiarray device. An optical microarray for the detection of toxic agents using a planar array of antibody probes was described by Ligler and coworkers [13]. Their system was composed of a CCD for detection, an excitation source and a microscope slide with a photoactivated optical adhesive. Antibodies against three different toxins, staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis, were covalently attached to small wells in the slide formed by the optical adhesive. The microscope slide was then mounted over the CCD with a gradient refractive index (GRIN) lens array used to focus the wells onto the CCD. Toxins were then introduced to the slide followed by Cy5-labeled antibodies. The bound antibodies were then excited and the resulting fluorescence from all of the sensor locations were monitored simultaneously. Concentrations ranging from 5-25 ng/mL were capable of being measured for the different toxins.

High-density oligonucleotide arrays, consisting of greater than 96 000 oligonucleotides have been designed by Hacia et al. for the screening of the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible variations in the homozygous and heterozygous states [35]. Single stranded RNA targets were created by PCR amplification followed by in vitro transcription and partial fragmentation. These targets were then tested and fluorescence responses from targets containing the four natural bases to greater than 5 592 different fully complimentary 25 mer oligonucleotide probes were found.

To examine the effect of uridine and adenosine on the hybridization specificity, 33 200 probes containing centrally localized base pair mismatches were constructed and tested. Targets that contained modified 5-methyluridine showed a localized enhancement in fluorescence hybridization signals. In general, oligonucleotide microarrays, often referred to as "DNA chips", are generally made by a light-directed chemical reaction that uses photographic masks for each chip [35]. A maskless fabrication method of light-directed oligonucleotide microarrays using a digital microarray has been reported [47]. In this method, a maskless array synthesizer replaces the chrome mask with virtual masks generated on a computer, which are relayed to a digital microarray.

1.3.2. Integrated Biochip Systems

The development of a truly integrated biochip having a phototransistor integrated circuit (IC) microchip has been reported by Vo-Dinh and coworkers [47, 48]. This work involves the integration of a 4 x 4 and 10 x 10 optical biosensor array onto an integrated circuit (Figure 1.5). Most optical biochip technologies are very large when the excitation source and detector are considered, making them impractical for anything but laboratory usage. In this biochip the sensors, amplifiers, discriminators and logic circuitry are all built onto the chip. In one biochip system, each of the sensing elements is composed of 220 individual phototransistor cells connected in parallel to improve the sensitivity of the instrument. The

FIGURE 1.5. Schematic diagram of an integrated biochip system with microchip sensor.

ability to integrate light emitting diodes (LEDs) as the excitation sources into the system is also discussed. An important element in the development of the multifunctional biochip (MFB) involves the design and development of an IC electro-optic system for the microchip detection elements using the complementary metal oxide silicon (CMOS) technology. With this technology, highly integrated biochips are made possible partly through the capability of fabricating multiple optical sensing elements and microelectronics on a single system. Applications of the biochip are illustrated by measurements of the HIV1 sequence-specific probes using the DNA biochip device for the detection of a gene segment of the AIDS virus [47]. Recently, a MFB which allows simultaneous detection of several disease endpoints using different bioreceptors, such as DNA, antibodies, enzymes, cellular probes, on a single biochip system was developed [22]. The MFB device was a self-contained system based on an integrated circuit including photodiode sensor arrays, electronics, amplifiers, discriminators and logic circuitry. The multi-functional capability of the MFB biochip device is illustrated by measurements of different types of bioreceptors using DNA probes specific to gene fragments of the Mycobacterium Tuberculosis (TB) system, and antibody probes targeted to the cancer related tumor suppressor gene p53.

A biochip equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassayshas been developed for E. coli detection [39]. The microfluidics system includes a reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. Studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.

A CMOS biochip coupled to multiplex capillary electrophoresis (CE) system has been developed [36, 37]. This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.

Antibody-immobilized capillary reactors coupled to biochip detection have been developed for E. coli O157:H7 detection using enzyme-linked immunosorbent assay (ELISA), and a biochip system [38]. ISA is very sensitive and selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system.

The detection limits of E. coli O157:H7 using ELISA and Cy5 label-based immunoassays were determined to be 3 cells and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.

The application of a biochip using the molecular beacon (MB) detection scheme has been reported [Culha et al, 2004]. The medical application of this biochip novel MB detection system for the analysis of the breast cancer gene BRCA1 was illustrated. The MB is designed for the BRCA1 gene and a miniature biochip system is used for detection. The detection of BRCA1 gene is successfully demonstrated in solution and the limit of detection (LOD) is estimated as 70 nM.

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