On-chip assay for determining the inhibitory effects and modes of action of drugs against xanthine oxidase.

Xanthine oxidase (XO) is a key enzyme that can catalyze the conversion of xanthine to uric acid, causing various diseases in humans. We have developed a high-throughput chip-based assay that uses a photodiode array (PDA) microchip system to explore the inhibitory effects of drug analogs on XO. Inhibitory activities of cyclosporin A, aminoglutethimide, dithranol and naringenin against XO were assessed using this chip-based xanthine assay in the presence or absence of the antioxidant enzyme, superoxide dismutase (SOD). In addition, the mechanism of drug action was also disclosed by monitoring the combined effect of respective drug analogs and SOD on XO in the assay. The assessment was based on the red light absorption property of nitroblue tetrazolium (NBT) formazan, formed by free radical-mediated NBT reduction. Compared to naringenin (50 and 100 microM; a known XO inhibitor), cyclosporin A (5 and 10 microM) exhibited similar XO inhibitory activity, whereas dithranol (1 and 3 microM) and aminoglutethimide (2.5 and 5mM) showed minimum XO inhibition. Low standard deviation obtained during the assay demonstrates the preciseness and accuracy of the developed approach. Compared to the existing methods, the developed approach is advantageous due to its simplicity and compatibility with high-throughput screening procedures. Furthermore, this approach can be applied to the early phase of drug discovery screening to explore various drug analogs for their XO inhibitory activities.

Development of a photosensitive, high-throughput chip-based superoxide dismutase (SOD) assay to explore the radioprotective activity of herbal plants.

Appropriate pharmacological interventions and modalities are needed to protect humans against the deleterious effects of ionizing radiation. We disclose a rapid chip-based approach to elucidate the radioprotective/antioxidant potential of herbal plants using a photodiode array (PDA) microchip system. Red light absorption property of nitroblue tetrazolium (NBT) formazan was applied to chip-based superoxide dismutase (SOD) activity measurements of six herbal plant extracts in a high-throughput manner. SOD activities obtained via gel-based assays were in line with the data obtained through the chip-based assay and hence validated our approach. Compared to amifostine, all the tested herbal plant extracts, except apricot kernel, demonstrated greater radioprotective properties. Among the tested herbal extracts, pueraria root showed the highest antioxidant/radioprotective activity and can be considered a preferred radioprotector candidate. Low standard deviations and high statistical confidence obtained during the assay prove the sensitivity and consistency of this approach. The developed approach has several advantages (simplicity, rapidness and portability) over existing methods and can be applied to high-throughput screening of the radioprotective properties of various unexplored plants species.

Interdigitated microelectrode array-coupled bipolar semiconductor photodiode array (IMEA-PDA) microchip for on-chip electrochemiluminescence detection.

This paper reports the design, fabrication and testing of a microchip wherein interdigitated microelectrode arrays (IMEA) were integrated with bipolar semiconductor photodiode array (PDA) chip to fabricate a highly compact embodiment for on-chip handling of solutions and electrochemiluminescence (ECL) detection. A 12 x 12 micro array of photodiodes, each coupled with an interdigitated microelectrode array (IMEA), an array of current amplifiers, and a photodiode element-addressing circuit were integrated into a single 2 x 2 cm² IC chip. Each photodiode had dimensions of 300 x 300 µm² and the photodiode-to-photodiode distance was 100 µm. The chip was successfully applied to the on-chip quantification of electro-chemiluminescing probe-labeled single stranded oligonucleotides. The minimum detectable limit at signal/noise ≥ 3 was found to be 5 x 10⁻¹4 moles of oligonucleotides with a sample volume as low as 5 microl (i.e., 10 fmole/µl). The attractive features of the developed IMEA-PDA microchip are that a plurality of samples can be analyzed simultaneously using a chip and that for a given sample the data can be averaged from values obtained from multiple, individually addressed pixels. These in turn bring in speed and statistical confidence in analysis. The IMEA-PDA microchip system has the potential to be used as a versatile and highly compact chemical analysis tool for chemical sensing and metrology applications.

Photodiode array on-chip biosensor for the detection of E. coli O157:H7 pathogenic bacteria.

An integrated circuit (IC) of photodiode array (PDA) microchip system was used for the on-chip detection of E. coli O157:H7 based on an enzymatic bioassay and light absorption property of the reaction product. The PDA microchip consisting of an array of 12 x 12 photodiode detection elements served as a photosensor as well as a protein-immobilizing sample platform. As a result, E. coli O157:H7 could be detected directly on the surface of PDA detection elements. E. coli O157:H7 was detected by forming a "sandwich-type" enzymatic immunocomplex on the PDA detection elements using an on-chip bioassay. The quantitative analysis of E. coli O157:H7 immunocomplex was carried out based on the light absorption property of the enzymatic reaction products of E. coli O157:H7 immunocomplexes with respect to a red beam produced by light emitting diodes (LEDs) installed right above the PDA microchip. During the on-chip bioassay, the wet photodiode detection elements exposed to a lot of biological materials or buffer solutions were capable of maintaining their photosensing capabilities. The portable PDA on-chip biosensor permits direct optical detection of E. coli O157:H7 and eliminates the necessity of the conventional expensive microplate reader that is incompatible with the size of the protein microarray.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus.

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

An automated method for in vitro anticancer drug efficacy monitoring based on cell viability measurement using a portable photodiode array chip.

An integrated circuit (IC) bipolar semiconductor photodiode array (PDA) microchip system coupled with light emitting diodes (LEDs) was used for rapid, automated cell viability measurements and high-throughput drug efficacy monitoring. Using the absorption property of trypan blue dye against the red light emitted by LEDs, we determined the effect of three anticancer drugs, viz., camptothecin (CAM), sodium salicylate (Na-Sal) and naringenin (Nar) on the cell viability of human promyelocytic leukemia cells (HL-60) and human embryonic kidney cells (HEK-293). Cell viabilities were measured based on the relative reduction in the photo responses of the photodiodes, covered with known concentration of trypan blue-stained cells. The developed method offers greater sensitivity and hence an excellent estimation of cell viability, but without all the hassle of conventional methods. Flow cytometric measurement and confocal microscopy were applied as complementary techniques for further validation of the results. The work presented here has important implications with regard to high-throughput measurement of optimal concentrations of different drugs against different cell lines in vitro.

Low noise bipolar photodiode array protein chip based on on-chip bioassay for the detection of E. coli O157:H7.

A bipolar photodiode array (PDA) protein chip is presented for the detection of E. coli O157:H7. Through unique design of the bipolar PDA microchip, the device was able to detect E. coli O157:H7 directly on the surface of the bipolar PDA. The bipolar PDA microchip maintained low noise level in the entire process of on-chip protein assay and demonstrated high performance of analog signal processing. At every reaction step of the on-chip bioassay, stability of wet photodiode detection elements was confirmed by monitoring the variance of their photosignals with respect to the irradiated red beam. The background signal represented less than 1.8% variance with respect to maximum signal of photodiode detection elements. As a result of using the on-chip bioassay, any complicated optical alignment and components could be removed in the constructed protein chip. This protein chip enables direct optical detection of E. coli O157:H7 eliminating the need of conventional expensive microplate reader that is incompatible with size of sampling platform of protein chip. The independence of the constructed protein chip on conventional microplate reader can contribute greatly to further miniaturization of protein chip and field usable lab-on-a-chip.