In vitro diagnosis of DNA methylation biomarkers with digital PCR in breast tumors.

Liquid biopsy of cancers using DNA methylation biomarkers has received significant interest, where the quantification of multiple biomarkers is generally needed for improving the sensitivity and specificity of cancer diagnosis. However, the inefficiency of the traditional quantitative polymerase chain reaction (qPCR)-based MethyLight assay for detecting the extremely low concentration of methylated DNA fragments in body fluids limits its clinical applications. Here, we developed an ultrasensitive microwell chip digital polymerase chain reaction (dPCR)-based MethyLight assay. Using the synthesized samples, the developed MethyLight assay can achieve 103-104-fold lower limit of detection and 1-16-fold lower limit of quantification than the traditional MethyLight assay. Four hypermethylated alleles (RARß2, BRCA1, GSTP1 and RASSF1A) related to breast cancer in twenty-three clinical samples were tested using the microwell chip dPCR-based MethyLight assay. The results showed that the dPCR assay achieves ∼2 times enhancement in the cancer detection rate over the traditional quantitative PCR. Furthermore, the dPCR can detect the healthy and benign samples, which are undetectable using the traditional MethyLight assay. In multiple gene analysis, we achieved the highest detection rate of 93.3% (in the "OR" format of RARß2 and GSTP1). Lastly, the estimated cut-off values in the dPCR assay were: <1, ∼1 to 100 and >100 (copies per µL) referring to the healthy, benign and malignant breast cancers, respectively. Therefore, the developed microwell chip dPCR-based MethyLight assay could provide a powerful tool for cancer biopsy diagnosis and disease monitoring.

A Controllable and Integrated Pump-enabled Microfluidic Chip and Its Application in Droplets Generating.

A microfluidic chip with a controllable and integrated piezoelectric pump was proposed and demonstrated, where the pump was designed as a micro-actuator based on polyvinylidene fluoride (PVDF) organic piezoelectric film. In this case, the pump should integrate with the microfluidics device very well into one chip. The flow rate can be precisely controlled in the range of 0-300 µl/min for water by tuning the Vpp and frequency of Alternating Current (AC) voltage applied on the diaphragm. To analyze the relationship between the flow rate and the deflection of diaphragm, the deformations of diaphragm at different voltages were researched. The displacement of diaphragm was defined as 17.2 µm at the voltages of 3.5 kV, 5 Hz when the pump chamber was full of water. We have used the integrated microfluidic chip with two pumps for droplet generation to demonstrate its great potential for application in droplet-based microfluidic chip.

Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.

Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR.

Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection.

Pathogenic bacteria cause serious harm to human health, which calls for the development of advanced detection methods. Herein, we developed a novel detection platform based on fluorescence resonance energy transfer (FRET) for rapid, ultrasensitive and specific bacteria detection, where gold nanoparticles (AuNPs, acceptor) were conjugated with aptamers while upconversion nanoparticles (UCNPs, donor) were functionalized with corresponding complementary DNA (cDNA). The spectral overlap between UCNPs fluorescence emission and AuNPs absorption enables the occurrence of FRET when hybridizing the targeted aptamer and cDNA, causing upconversion fluorescence quenching. In the presence of target bacteria, the aptamers preferentially bind to bacteria forming a three-dimensional structure and thereby dissociate UCNPs-cDNA from AuNPs-aptamers, resulting in the recovery of upconversion fluorescence. Using the UCNPs based FRET aptasensor, we successfully detected Escherichia coli ATCC 8739 (as a model analyte) with a detection range of 5-106cfu/mL and detection limit of 3cfu/mL. The aptasensor was further used to detect E. coli in real food and water samples (e.g., tap/pond water, milk) within 20min. The novel UCNPs based FRET aptasensor could be used to detect a broad range of targets from whole cells to metal ions by using different aptamer sequences, holding great potential in environmental monitoring, medical diagnostics and food safety analysis.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 µg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications.

Portable microfluidic and smartphone-based devices for monitoring of cardiovascular diseases at the point of care.

Cardiovascular diseases (CVDs) are the main causes of morbidity and mortality in the world where about 4 in every 5 CVD deaths happen in low- and middle-income countries (LMICs). Most CVDs are preventable and curable, which is largely dependent on timely and effective interventions, including diagnosis, prognosis and therapeutic monitoring. However, these interventions are high-cost in high income countries and are usually lacking in LMICs. Thanks to the rapid development of microfluidics and nanotechnology, lots of portable analytical devices are developed for detection of CVDs at the point-of-care (POC). In the meantime, smartphone, as a versatile and powerful handheld tool, has been employed not only as a reader for microfluidic assays, but also as an analyzer for physiological indexes. In this review, we present a comprehensive introduction of the current status and potential development direction on POC diagnostics for CVDs. First of all, we introduce some main facts about CVDs and their standard diagnostic procedures and methods. Second, we discuss about both commercially available POC devices and developed prototypes for detection of CVDs via immunoassays. Subsequently, we report the advances in smartphone-based readout for microfluidic assays. Finally, we present some examples using smartphone, individually or combined with other components or devices, for CVD monitoring. We envision an integrated smartphone-based system capable of functioning blood tests, disease examination, and imaging will come in the future.

The potential health challenges of TiO2 nanomaterials.

Titanium dioxide (TiO2 ) nanomaterials (NMs) have found widespread applications owing to their attractive physical and chemical properties. As a result, the potential adverse impacts of nano-TiO2 exposure on humans have become a matter of concern. This review presents the state-of-the-art advances on the investigations of the adverse effects of NMs, including the potential exposure routes of nano-TiO2 (e.g. respiratory system, skin absorption and digestive system), the physico-chemical characterizations of nano-TiO2 (e.g. crystal structure, shape,size, zeta potential, treatment media, aggregation and agglomeration tendency, surface characteristics and coatings), risk evaluation of nanotoxicity (e.g. cytotoxicity, ecotoxicity, phototoxicity, and phytotoxicity) and potential mechanisms of adverse effects (e.g. generation of reactive oxygen species, oxidative stress and organelle dysfunction). The review aims to facilitate scientific assessments of health risks to nano-TiO2 , which would guide the safe applications of NMs in our daily life.