An interferometric imaging biosensor using weighted spectrum analysis to confirm DNA monolayer films with attogram sensitivity.

Interferometric imaging biosensors are powerful and convenient tools for confirming the existence of DNA monolayer films on silicon microarray platforms. However, their accuracy and sensitivity need further improvement because DNA molecules contribute to an inconspicuous interferometric signal both in thickness and size. Such weaknesses result in poor performance of these biosensors for low DNA content analyses and point mutation tests. In this paper, an interferometric imaging biosensor with weighted spectrum analysis is presented to confirm DNA monolayer films. The interferometric signal of DNA molecules can be extracted and then quantitative detection results for DNA microarrays can be reconstructed. With the proposed strategy, the relative error of thickness detection was reduced from 88.94% to merely 4.15%. The mass sensitivity per unit area of the proposed biosensor reached 20 attograms (ag). Therefore, the sample consumption per unit area of the target DNA content was only 62.5 zeptomoles (zm), with the volume of 0.25 picolitres (pL). Compared with the fluorescence resonance energy transfer (FRET), the measurement veracity of the interferometric imaging biosensor with weighted spectrum analysis is free to the changes in spotting concentration and DNA length. The detection range was more than 1µm. Moreover, single nucleotide mismatch could be pointed out combined with specific DNA ligation. A mutation experiment for lung cancer detection proved the high selectivity and accurate analysis capability of the presented biosensor.

A fully integrated microchip system for automated forensic short tandem repeat analysis.

We have successfully developed an integrated microsystem that combines two plastic microchips for DNA extraction and PCR amplification with a glass capillary array electrophoresis chip together in a compact control and detection instrument for automated forensic short tandem repeat (STR) analysis. DNA extraction followed by an "in situ PCR" was conducted in a single reaction chamber of the microchip based on a filter paper-based extraction methodology. PCR products were then mixed with sizing standards by an injection electrode and injected into the electrophoresis chip for four-color confocal fluorescence detection. The entire STR analysis can be completed in about two hours without any human intervention. Since the 15-plex STR system has a more stringent requirement for PCR efficiency, we optimized the structure of the plastic DNA extraction and amplification chip, in which the reaction chamber was formed by sandwiching a hollow structure layer with two blank cover layers, to reduce the adsorption of PCR reagents to the surfaces. In addition, PCR additives, bovine serum albumin, poly(ethylene glycol), and more magnesium chloride were included into the on-chip multiplex STR system. The limit-of-detection study demonstrated that our microsystem was able to produce full 15-plex STR profiles from 3.75 ng standard K562 DNA. Buccal swab and whole blood samples were also successfully typed by our system, validating the feasibility of performing rapid DNA typing in a "sample-in-answer-out" manner for on-site forensic human identification.

Label-Free Method Using a Weighted-Phase Algorithm To Quantitate Nanoscale Interactions between Molecules on DNA Microarrays.

White light interference is used as a label-free method to detect nanoscale changes on surfaces. However, the signal-to-noise ratio of the white light interference method is very low, thus resulting in inaccurate results. In this paper, we report a corrected label-free method based on hyperspectral interferometry to overcome the shortcoming of the white light interference method. A platform based on hyperspectral interferometry was established, and a DNA hybridization microarray was constructed to quantitate thickness variation of molecules on a solid surface. We used fluorescence resonance energy transfer (FRET) to validate the results of our method. Compared to conventional fluorescence-labeled method like FRET, our method has advantages because it does not require a fluorescent label and has a detection limit of 1.78 nm, a high accuracy, and wide detection range (5-64 bp).

Chitosan-Modified Filter Paper for Nucleic Acid Extraction and "in Situ PCR" on a Thermoplastic Microchip.

Plastic microfluidic devices with embedded chitosan-modified Fusion 5 filter paper (unmodified one purchased from GE Healthcare) have been successfully developed for DNA extraction and concentration, utilizing two different mechanisms for DNA capture: the physical entanglement of long-chain DNA molecules with the fiber matrix of the filter paper and the electrostatic adsorption of DNA to the chitosan-modified filter fibers. This new method not only provided a high DNA extraction efficiency at a pH of 5 by synergistically combining these two capture mechanisms together, but also resisted the elution of DNA from filters at a pH > 8 due to the entanglement of DNA with fibers. As a result, PCR buffers can be directly loaded into the extraction chamber for "in situ PCR", in which the captured DNA were used for downstream analysis without any loss. We demonstrated that the capture efficiencies of a 3-mm-diameter filter disc in a microchip were 98% and 95% for K562 human genomic DNA and bacteriophage λ-DNA, respectively. The washes with DI water, PCR mixture, and TE buffer cannot elute the captured DNA. In addition, the filter disc can enrich 62% of λ-DNA from a diluted sample (0.05 ng/µL), providing a concentration factor more than 30-fold. Finally, a microdevice with a simple two-chamber structure was developed for on-chip cell lysis, DNA extraction, and 15-plex short tandem repeat amplification from blood. This DNA extraction coupled with "in situ PCR" has great potential to be utilized in fully integrated microsystems for rapid, near-patient nucleic acid testing.

A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms.

A fully integrated and automated microsystem consisting of low-cost, disposable plastic chips for DNA extraction and PCR amplification combined with a reusable glass capillary array electrophoresis chip in a modular-based format was successfully developed for warfarin pharmacogenetic testing. DNA extraction was performed by adopting a filter paper-based method, followed by "in situ" PCR that was carried out directly in the same reaction chamber of the chip without elution. PCR products were then co-injected with sizing standards into separation channels for detection using a novel injection electrode. The entire process was automatically conducted on a custom-made compact control and detection instrument. The limit of detection of the microsystem for the singleplex amplification of amelogenin was determined to be 0.625 ng of standard K562 DNA and 0.3 µL of human whole blood. A two-color multiplex allele-specific PCR assay for detecting the warfarin-related single-nucleotide polymorphisms (SNPs) 6853 (-1639G>A) and 6484 (1173C>T) in the VKORC1 gene and the *3 SNP (1075A>C) in the CYP2C9 gene was developed and used for validation studies. The fully automated genetic analysis was completed in two hours with a minimum requirement of 0.5 µL of input blood. Samples from patients with different genotypes were all accurately analyzed. In addition, both dried bloodstains and oral swabs were successfully processed by the microsystem with a simple modification to the DNA extraction and amplification chip. The successful development and operation of this microsystem establish the feasibility of rapid warfarin pharmacogenetic testing in routine clinical practice.

An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis.

We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-µl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, "amplicon-in-answer-out" operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis.

Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-µL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-µL human whole blood followed by amplifications of 59-bp ß-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 µL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 µL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 µm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

Sensitive and high resolution subcutaneous fluorescence in vivo imaging using upconversion nanoparticles and microarrays.

A sensitive and high resolution small animal in vivo imaging system using upconversion nanoparticles (UNPs) and microarrays was developed. The fluorescence tomography using UNPs could achieve higher precision than that using ordinary fluorophores, which was theoretically explained by the finite element method (FEM). Given the autofluorescence-insensitive property of UNPs, a high subcutaneous detection sensitivity of 0.93 × 10(-4) wt% could be achieved with a UNP volume of ∼10 µL in tissue phantoms. Furthermore, UNP fluorophore microarrays (25, 50 and 100 µm arrays) embedded under mouse skin were prepared for subcutaneous in vivo detection. An optical clearing method was applied to enhance the skin transparency and improve the spatial resolution. The results demonstrated that the optimized system could achieve a spatial resolution of 50 µm for in vivo detection of subcutaneous UNP microarrays. Taken together, we conclude that the proposed system and UNP microarrays could achieve sensitive, high resolution subcutaneous in vivo detection, and have great potential for high throughput detection of tumors and other diseases.