One-Step RT-qPCR for Viral RNA Detection Using Digital Analysis.

The rapid detection of viruses is becoming increasingly important to prevent widespread infections. However, virus detection via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is time-consuming, as it involves independent nucleic acid extraction and complementary DNA synthesis. This process limits the potential for rapid diagnosis and mass analysis, which are necessary to curtail viral spread. In this study, a simple and rapid thermolysis method was developed to circumvent the need for extraction and purification of viral RNA. The developed protocol was applied to one-chip digital PCR (OCdPCR), which allowed thermolysis, RT, and digital PCR in a single unit comprising 20,000 chambers of sub-nanoliter volume. Two viruses such as tobacco mosaic virus and cucumber mosaic virus were tested as model viral particles. First, the temperature, exposure time, and template concentration were optimized against tobacco mosaic viral particles, and the most efficient conditions were identified as 85°C, 5 min, and 0.01 µg/nL with a cycle threshold of approximately 33. Finally, the OCdPCR analysis yielded 1,130.2 copies/µL using 10-2 µg/nL of viral particles in a 30 min thermolysis-RT reaction at 70°C. This novel protocol shows promise as a quick, accurate, and precise method for large-scale viral analysis in the future.

Surface Activity-Tuned Metal Oxide Chemiresistor: Toward Direct and Quantitative Halitosis Diagnosis.

Continuous monitoring of hydrogen sulfide (H2S) in human breath for early stage diagnosis of halitosis is of great significance for prevention of dental diseases. However, fabrication of a highly selective and sensitive H2S gas sensor material still remains a challenge, and direct analysis of real breath samples has not been properly attempted, to the best of our knowledge. To address the issue, herein, we introduce facile cofunctionalization of WO3 nanofibers with alkaline metal (Na) and noble metal (Pt) catalysts via the simple addition of sodium chloride (NaCl) and Pt nanoparticles (NPs), followed by electrospinning process. The Na-doping and Pt NPs decoration in WO3 grains induces the partial evolution of the Na2W4O13 phase, causing the buildup of Pt/Na2W4O13/WO3 multi-interface heterojunctions that selectively interacts with sulfur-containing species. As a result, we achieved the highest-ranked sensing performances, that is, response (Rair/Rgas) = 780 @ 1 ppm and selectivity (RH2S/REtOH) = 277 against 1 ppm ethanol, among the chemiresistor-based H2S sensors, owing to the synergistic chemical and electronic sensitization effects of the Pt NP/Na compound cocatalysts. The as-prepared sensing layer was proven to be practically effective for direct, and quantitative halitosis analysis based on the correlation (accuracy = 86.3%) between the H2S concentration measured using the direct breath signals obtained by our test device (80 cases) and gas chromatography. This study offers possibilities for direct, highly reliable and rapid detection of H2S in real human breath without the need of any collection or filtering equipment.

Wrist-wearable bioelectrical impedance analyzer with miniature electrodes for daily obesity management.

Bioelectrical impedance analysis (BIA) is used to analyze human body composition by applying a small alternating current through the body and measuring the impedance. The smaller the electrode of a BIA device, the larger the impedance measurement error due to the contact resistance between the electrode and human skin. Therefore, most commercial BIA devices utilize electrodes that are large enough (i.e., 4 × 1400 mm2) to counteract the contact resistance effect. We propose a novel method of compensating for contact resistance by performing 4-point and 2-point measurements alternately such that body impedance can be accurately estimated even with considerably smaller electrodes (outer electrodes: 68 mm2; inner electrodes: 128 mm2). Additionally, we report the use of a wrist-wearable BIA device with single-finger contact measurement and clinical test results from 203 participants at Seoul St. Mary's Hospital. The correlation coefficient and standard error of estimate of percentage body fat were 0.899 and 3.76%, respectively, in comparison with dual-energy X-ray absorptiometry. This result exceeds the performance level of the commercial upper-body portable body fat analyzer (Omron HBF-306). With a measurement time of 7 s, this sensor technology is expected to provide a new possibility of a wearable bioelectrical impedance analyzer, toward obesity management.

Toward a disposable low-cost LOC device: heterogeneous polymer micro valve and pump fabricated by UV/ozone-assisted thermal fusion bonding.

Herein, a heterogeneous polymer micro valve and pump with a polypropylene (PP) membrane was developed in a low-cost manner via UV/ozone-assisted thermal fusion bonding. The proposed fabrication technique allowed for a geometrically selective bonding; consequently, the membrane was prevented from bonding with the valve seat of the diaphragm micro-valve, without patterning a protection layer or introducing an additional structure. The developed device withstands 480 kPa of static pressure and up to 350 kPa of a vibration pressure, providing sufficient bonding strength for microfluidic actuations. The fabricated micro valve and pump are fully characterized and compared with a poly(dimethylsiloxane) (PDMS) membrane glass device, showing comparable valving and pumping performance. As a result, the robust PP membrane micro valve and pump are simply implemented in a facile manner, and demonstrated excellent performance, which is highly desirable for mass production of disposable lab-on-a-chip (LOC) devices.

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs.

Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs.

We have developed a bead-packed microfluidic device with a built-in flexible wall to automate extraction of nucleic acids from methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs. The flexible polydimethylsiloxane (PDMS) membrane was designed to manipulate the surface-to-volume ratio (SVR) of bead-packed chambers in the range of 0.05 to 0.15 (µm(-1)) for a typical solid phase extraction protocol composed of binding, washing, and eluting. In particular, the pneumatically assisted close packing of beads led to an invariant SVR (0.15 µm(-1)) even with different bead amounts (10-16 mg), which allowed for consistent operation of the device and improved capture efficiency for bacteria cells. Furthermore, vigorous mixing by asynchronous membrane vibration enabled ca. 90% DNA recovery with ca. 10 µL of liquid solution from the captured cells on the bead surfaces. The full processes to detect MRSA in nasal swabs, i.e., nasal swab collection, prefiltration, on-chip DNA extraction, and real-time polymerase chain reaction (PCR) amplification, were successfully constructed and carried out to validate the capability to detect MRSA in nasal swab samples. This flexible microdevice provided an excellent analytical PCR detection sensitivity of ca. 61 CFU/swab with 95% confidence interval, which turned out to be higher than or similar to that of the commercial DNA-based MRSA detection techniques. This excellent performance would be attributed to the capability of the flexible bead-packed microdevice to enrich the analyte from a large initial sample (e.g., 1 mL) into a microscale volume of eluate (e.g., 10 µL). The proposed microdevice will find many applications as a solid phase extraction method toward various sample-to-answer systems.

Functional integration of DNA purification and concentration into a real time micro-PCR chip.

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.

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.

Nanopore sensor for fast label-free detection of short double-stranded DNAs.

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with gamma-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.

Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus.

The polymerase chain reaction (PCR) is widely used to amplify a small amount of DNA in samples for genetic analysis. Rapid and accurate amplification is prerequisite for broad applications including molecular diagnostics of diseases, food safety, and biological warfare tests. We have developed a rapid real-time micro-scale chip-based PCR system, which consists of six individual thermal cycling modules capable of independent control of PCR protocols. The PCR volume is 1 microl and it takes less than 20 min to complete 40 thermal cycles. To test utility of a chip-based PCR system as a molecular diagnostic device, we have conducted the first large-scale clinical evaluation study. Three independent clinical evaluation studies (n = 563) for screening the hepatitis B virus (HBV) infection, the most popular social epidemic disease in Asia, showed an excellent sensitivity, e.g. 94%, and specificity, e.g. 93%, demonstrating micro-scale chip-based PCR can be applied in molecular diagnostics.

World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays.

We report a practical world-to-chip microfluidic interfacing method with built-in valves suitable for microscale multichamber chip-based assays. One of the primary challenges associated with the successful commercialization of fully integrated microfluidic systems has been the lack of reliable world-to-chip microfluidic interconnections. After sample loading and sealing, leakage tests were conducted at 100 degrees C for 30 min and no detectable leakage flows were found during the test for 100 microchambers. To demonstrate the utility of our world-to-chip microfluidic interface, we designed a microscale PCR chip with four chambers and performed PCR assays. The PCR results yielded a 100% success rate with no contamination or leakage failures. In conclusion, we have introduced a simple and inexpensive microfluidic interfacing system for both sample loading and sealing with no dead volume, no leakage flow and biochemical compatibility.