Christopher Ko

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood utilizing centrifugal microfluidics on a polymer based CD platform. By combining the TS-LIMBS (target separation and laser-irradiated magnetic bead system) and centrifugal microfluidics using the novel LIFM (the laser irradiated ferrowax microvalves), DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) were conducted. The total process was finished within 12 minutes with only one manual step of loading 100 muL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD was as good as that of the samples prepared in conventional bench top method.

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

Prediction of Recurrence-Free Survival in Postoperative Non–Small Cell Lung Cancer Patients by Using an Integrated Model of Clinical Information and Gene Expression

Purpose: One of the main challenges of lung cancer research is identifying patients at high risk for recurrence after surgical resection. Simple, accurate, and reproducible methods of evaluating individual risks of recurrence are needed. Experimental Design: Based on a combined analysis of time-to-recurrence data, censoring information, and microarray data from a set of 138 patients, we selected statistically significant genes thought to be predictive of disease recurrence. The number of genes was further reduced by eliminating those whose expression levels were not reproducible by real-time quantitative PCR. Within these variables, a recurrence prediction model was constructed using Cox proportional hazard regression and validated via two independent cohorts (n = 56 and n = 59). Results: After performing a log-rank test of the microarray data and successively selecting genes based on real-time quantitative PCR analysis, the most significant 18 genes had P values of <0.05. After subsequent stepwise variable selection based on gene expression information and clinical variables, the recurrence prediction model consisted of six genes (CALB1, MMP7, SLC1A7, GSTA1, CCL19, and IFI44). Two pathologic variables, pStage and cellular differentiation, were developed. Validation by two independent cohorts confirmed that the proposed model is significantly accurate (P = 0.0314 and 0.0305, respectively). The predicted median recurrence-free survival times for each patient correlated well with the actual data. Conclusions: We have developed an accurate, technically simple, and reproducible method for predicting individual recurrence risks. This model would potentially be useful in developing customized strategies for managing lung cancer.

Bacteria concentration using a membrane type insulator-based dielectrophoresis in a plastic chip

We report an insulator‐based (or, electrodeless) dielectrophoresis utilizing microfabricated plastic membranes. The membranes with honeycomb‐type pores have been fabricated by patterning the SU‐8 layer on a substrate which was pretreated with self‐assembled monolayer of octadecyltrichlorosilane for the easy release. The fabricated membrane was positioned between two electrodes and alternating current field was applied for the particle trap experiments. The particle could be trapped due to the dielectrophoresis force generated by the non‐uniformities of the electric fields applied through the membranes with pores. Simulations using CFD‐ACE+(CFD Research, Huntsville, Alabama) suggested that the dielectrophoresis force is stronger in the edge of the pores where the field gradient is highest. The bacteria could be captured on the near edge of the pores when the electric field was turned on and the trapped bacteria could be released when the field was turned off with the release efficiency of more than 93±7%. The maximal trapping efficiency of 66±7% was obtained under the electric fields (E=128 V/mm and f=300 kHz) when the dilute bacteria solution (Escherichia coli: 9.3×103 cell/mL, 0.5 mS/m) flowed with a flow rate of 100 μL/min.

Surface acoustic wave immunosensor for real-time detection of hepatitis B surface antibodies in whole blood samples

We demonstrate an application of Love wave mode surface acoustic wave (SAW) immunosensor to detect hepatitis B surface antibody (HBsAb) in aqueous conditions. SiO(2) guiding layer was deposited on 36 degrees YX-LiTaO(3) piezoelectric single crystal substrate to protect the electrodes and to trap the acoustic energy near the surface, and hepatitis B surface antigen (HBsAg) was immobilized on the sensing area. The resonance frequency shift was monitored to detect specific binding of HBsAb to immobilized HBsAg. To eliminate the effects of other physical factors except for the mass change, the resonance frequency was compared to that of a reference SAW device coated with bovine serum albumin (BSA) to block binding of HBsAb. The guiding layer thickness with maximum mass sensitivity was found to be 5 microm, which was in agreement with the theoretical calculation, and the center resonance frequency was around 199 MHz. The sensor showed binding specificity to HBsAb and a linear relationship between the frequency shift and the antibody concentration with sensitivity of 0.74 Hz/(pg/microl) and detection limit below 10 pg/microl. In addition, our SAW immunosensor successfully detected HBsAb in whole blood samples without any pretreatment, opening up its applicability in fast label-free protein detection methods.

Bacterial DNA Sample Preparation from Whole Blood Using Surface-Modified Si Pillar Arrays

A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip.

Label-Free CMOS DNA Quantification with On-Chip Noise Reduction Schemes

We present a label-free CMOS DNA sensor with a new sensing-pixel architecture and background noise reduction scheme. The proposed sensor generates a differential signal between bio-samples and reference buffer solution with significant reduction in offset and gain fixed pattern noise by employing on-chip correlated double sampling circuits. Non-surface-binding detection technique allows to quantify DNA molecules continuously and sequentially and to reuse the sensor by simple washing protocol. By directly reading the negative charges of DNA molecules, DNA concentrations from 1 muM to 10 muM have been successfully discriminated.

One-Step Pathogen Specific DNA Extraction from Whole Blood on a Centrifugal Microfluidic Device

We could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood utilizing centrifugal microfluidics on a polymer based CD platform. By combining the TS-LIMBS (target separation and laser-irradiated magnetic bead system) and centrifugal microfluidics using the novel LIFM (the laser irradiated ferrowax microvalves), DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) were conducted. The total process was finished within 12 minutes with only one manual step of loading 100 muL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD was as good as that of the samples prepared in conventional bench top method.

Real-time label-free quantitative monitoring of biomolecules without surface binding by floating-gate complementary metal-oxide semiconductor sensor array integrated with readout circuitry

We report a label-free field-effect sensing array integrated with complementary metal-oxide semiconductor (CMOS) readout circuitry to detect the surface potential determined by the negative charge in DNA molecules. For real-time DNA quantification, we have demonstrated the measurements of DNA molecules without immobilizing them on the sensing surface which is composed of an array of floating-gate CMOS transistors. This nonimmobilizing technique allows the continuous monitoring of the amount of charged molecules by injecting DNA solutions sequentially. We have carried out the real-time quantitative measurement of 19bp oligonucleotides and analyzed its sensitivity as a function of pH in buffer solutions.

SAW immunosensors for HBsAb detection

This paper presents the implementation of a surface acoustic wave (SAW) immunosensor system for real sample detection of antibody to hepatitis B surface antigen (HBsAg). The SAW sensor device was based on the mass detection using LOVE wave with the central frequency of 200 MHz. The thickness of the SiO2 guiding layer was optimized both theoretically and experimentally to obtain the maximum sensitivity. A detection circuitry using a differential scheme measured the frequency difference by subtracting the resonant frequency of the sample SAW from that of the reference SAW. The sensitivity of the sensor was 0.74 Hz/(pg/mulscr) and the detection limit was 10 pg/mulscr. Our SAW sensor successfully detected natural anti-HBsAg antidbody (HBsAb) of the real whole blood samples, which opens up a possibility of label-free protein detection without any pretreatment procedures.