Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species.

Panax ginseng and Panax quinquefolius have different medicinal properties and market values; however, they can be difficult to distinguish from one another based on physical appearances alone. Therefore, a molecular test that can be performed in commercial settings is needed to overcome this difficulty. A locus that contains a single nucleotide polymorphism (SNP) site to differentiate between P. ginseng and P. quinquefolius has been selected. An isothermal nucleic acid amplification test (NAAT) has been developed for use in a microfluidic chip; this NAAT method, which is based on lesion-induced DNA amplification (LIDA), amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT method was used to authenticate five ginseng root samples which indicated that two of the five samples appear to be mislabeled. These authentication results were consistent with those obtained from next generation sequencing (NGS) although this molecular test is more affordable and faster than NGS.

Centrifugal dynamic hybridization conducted in a microfluidic chip for signal enhancement in nucleic acid tests.

A rotating platform has been developed using a centrifugal chip holder to mount the standard chips for liquid delivery achieved by centrifugal pumping. This platform allows for dynamic hybridization to be performed in the microchannels constructed in the standard chips made using the 50 mm × 75 mm glass slides which allows for fast hybridization reactions. The results show that when the oligonucleotide-oligonucleotide hybridization is performed, there is good differentiation between perfectly complementary strands over 1 bp-mismatching counterparts when the long target strand is firstly immobilized and the short probe is secondly hybridized (Method 2), but not when the short probe is first immobilized, and the long target is subsequently hybridized (Method 1). When the differentiation between immobilized ginseng PCR product strands is performed, the correct result is achievable by Method 1, after signal enhancement and addition of formamide. The use of Method 2 is successful only when the PCR strand is captured, but not immobilized. In both methods, proper differentiation is achievable using the N1Q probe, un-achievable without centrifugal hybridization.

The genetic authentication of Panax ginseng and Panax quinquefolius based on using single nucleotide polymorphism (SNP) conducted in a nucleic acid test chip.

Panax ginseng and Panax quinquefolius, which are commonly called Chinese ginseng and American ginseng respectively, have different medicinal properties and market values; however, these samples can be difficult to differentiate from one another based on physical appearances of the samples especially when they are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this technique is based on the nucleic acid test (NAT) conducted on the microfluidic chip surface. Three single nucleotide polymorphism (SNP) sites (i.e. N1, N2, N3) on the Panax genome that differ between P. ginseng (G) and P. quinquefolius (Q) have been selected to design probes for the NAT. Primers were designed to amplify the antisense strands by asymmetric PCR. We have developed three different NAT methodologies involving surface immobilization and subsequent (stop flow or dynamic) hybridization of probes (i.e. N1G, N1Q, N2G, N2Q, N3Q) to the antisense strands. These NAT methods consist of two steps, namely immobilization and hybridization, and each method is distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand). These three NATs developed are called probe-target method 1, target-probe method 2 and three-strand complex method 3. Out of the three methods, it was found that the capture strand-target-probe method 3 provided the best differentiation of the ginseng species, in which a 3' NH2 capture strand is first immobilized and the antisense PCR strand is then bound, while N2G and N3Q probes are used for detection of P. ginseng (G) and P. quinquefolius (Q) respectively.

Screening of high-efficiency and low-toxicity antitumor active components in Macleaya cordata seeds based on the competitive effect of drugs on double targets by a new laminar flow chip.

It is urgent to obtain targeted drugs that selectively bind to pathological targets rather than physiological targets in the early stage of drug screening. G-Quadruplex has become one of the important targets in the development of anti-tumor drugs. However, drugs that target quadruplexes may also bind to dsDNA, which may lead to adverse reactions. In this study, a new three-phase laminar flow chip was constructed to enable the multi-components of a traditional Chinese medicine extract to dynamically and competitively bind with G-quadruplex DNA (on target) and double-stranded DNA (off target), so as to select high-efficiency and low-toxicity anti-tumor drugs. The results showed that there were five compounds in the extracts of Macleaya cordata seeds that exhibited obvious differences in binding to the two targets. Furthermore, the binding constants and modes of four identified alkaloids as they bound to two DNA targets were verified by fluorescence spectra and molecular docking methods. The toxicity to HepG2 and LO2 cells from the four alkaloids was also compared. The results showed that sanguinarine and chelerythrine could be used as candidate drugs with stronger binding to HT24 than DNA26. The chip can also be used for other types of double-target screening of other traditional Chinese medicine extracts or compound libraries.

Presence of an EML4-ALK gene fusion detected by microfluidic chip DNA hybridization.

Non-small cell lung cancer (NSCLC) accounts for ∼80-85% of all lung cancer cases, and the EML4-ALK fusion oncogene is a well-known contributor to NSCLC cases. Expensive methods such as FISH, IHC, and NGS have been used to detect the EML4-ALK fusion oncogene. Here, a cost-effective and facile method of detecting and differentiating an EML4-ALK fusion oncogene from the wild-type gene has been accomplished by DNA hybridization using the microfluidic biochip. First, oligonucleotide probes were confirmed for successful detection of immobilized sense strands. Second, capture of the sense PCR product strands (fusion and WT) and their subsequent detection and differentiation were accomplished. Our proof-of-concept study shows the ability to detect 1% fusion products, among WT ones.

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems.

Here, we report a microfluidic same-single-cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single-cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P-glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control on both fluorescence detection systems due to the inhibition by CsA.

Dielectrophoretic Microfluidic Chip Enables Single-Cell Measurements for Multidrug Resistance in Heterogeneous Acute Myeloid Leukemia Patient Samples.

The front-line treatment for adult acute myeloid leukemia (AML) is anthracycline-based combination chemotherapy. However, treatment outcomes remain suboptimal with relapses frequently observed. Among the mechanisms of treatment failure is multidrug resistance (MDR) mediated by the ABCB1, ABCC1, and ABCG2 drug-efflux transporters. Although genetic and phenotypic heterogeneity between leukemic blast cells is a well-recognized phenomenon, there remains minimal data on differences in MDR activity at the individual cell level. Specifically, functional assays that can distinguish the variability in MDR activity between individual leukemic blasts are lacking. Here, we outline a new dielectrophoretic (DEP) chip-based assay. This assay permits measurement of drug accumulation in single cells, termed same-single-cell analysis in the accumulation mode (SASCA-A). Initially, the assay was optimized in pretherapy samples from 20 adults with AML whose leukemic blasts had MDR activity against the anthracyline daunorubicin (DNR) tested using multiple MDR inhibitors. Parameters tested were initial drug accumulation, time to achieve signal saturation, fold-increase of DNR accumulation with MDR inhibition, ease of cell trapping, and ease of maintaining the trapped cells stationary. This enabled categorization into leukemic blast cells with MDR activity (MDR(+)) and leukemic blast cells without MDR activity (MDR(-ve)). Leukemic blasts could also be distinguished from benign white blood cells (notably these also lacked MDR activity). MDR(-ve) blasts were observed to be enriched in samples taken from patients who went on to enter complete remission (CR), whereas MDR(+) blasts were frequently observed in patients who failed to achieve CR following front-line chemotherapy. However, pronounced variability in functional MDR activity between leukemic blasts was observed, with MDR(+) cells not infrequently seen in some patients that went on to achieve CR. Next, we tested MDR activity in two paired AML patient samples. Pretherapy samples taken from patients that achieved CR to front-line chemotherapy were compared with samples taken at time of subsequent relapse. MDR(+) cells were frequently observed in leukemic blast cells in both pretherapy and relapsed samples, consistent with MDR as a mechanism of relapse in these patients. We demonstrate the ability of a new DEP microfluidic chip-based assay to identify heterogeneity in MDR activity in leukemic blasts. The test provides a platform for future studies to characterize the mechanistic basis for heterogeneity in MDR activity at the individual cell level.

Kras gene codon 12 mutation detection enabled by gold nanoparticles conducted in a nanobioarray chip.

This study employs a nanobioarray (NBA) chip for multiple biodetection of single base pair mutations at the Kras gene codon 12. To distinguish between the mutant and wild-type target DNAs, current bioarray methods use high-temperature hybridization of the targets to the allele-specific probes. However, these techniques need prior temperature optimization and become harder to implement in the case of the detection of multiple mutations. We aimed to detect these mutations at a single temperature (room temperature), enabled by the use of gold nanoparticles (AuNPs) on the bioarray created within nanofluidic channels. In this method, a low amount of target oligonucleotides (5fmol) and polymerase chain reaction (PCR) products (300pg) were first loaded on the AuNP surface, and then these AuNP-bound targets were introduced into the channels of a polydimethylsiloxane (PDMS) glass chip. The targets hybridized to their complementary probes at the intersection of the target channels to the pre-printed oligonucleotide probe lines on the glass surface, creating a bioarray. Using this technique, fast and high-throughput multiple discrimination of the Kras gene codon 12 were achieved at room temperature using the NBA chip, and the specificity of the method was proved to be as high as that with the temperature stringency method.

Same-single-cell analysis using the microfluidic biochip to reveal drug accumulation enhancement by an amphiphilic diblock copolymer drug formulation.

Multidrug resistance (MDR) is one of the major obstacles in drug delivery, and it is usually responsible for unsuccessful cancer treatment. MDR may be overcome by using MDR inhibitors. Among different classes of these inhibitors that block drug efflux mediated by permeability-glycoprotein (P-gp), less toxic amphiphilic diblock copolymers composed of methoxypolyethyleneglycol-block-polycaprolactone (MePEG-b-PCL) have been studied extensively. The purpose of this work is to evaluate how these copolymer molecules can reduce the efflux, thereby enhancing the accumulation of P-gp substrates (e.g., daunorubicin or DNR) in MDR cells. Using conventional methods, it was found that the low-molecular-weight diblock copolymer, MePEG17-b-PCL5 (PCL5), enhanced drug accumulation in MDCKII-MDR1 cells, but the high-molecular-weight version, MePEG114-b-PCL200 (PCL200), did not. However, when PCL200 was mixed with PCL5 (and DNR) in order to encapsulate them to facilitate drug delivery, there was no drug enhancement effect attributable to PCL5, and the reason for this negative result was unclear. Since drug accumulation measured on different cell batches originated from single cells, we employed the same-single-cell analysis in the accumulation mode (SASCA-A) to find out the reason. A microfluidic biochip was used to select single MDR cells, and the accumulation of DNR was fluorescently measured in real time on these cells in the absence and presence of PCL5. The SASCA-A method allowed us to obtain drug accumulation information faster in comparison to conventional assays. The SASCA-A results, and subsequent curve-fitting analysis of the data, have confirmed that when PCL5 was encapsulated in PCL200 nanoparticles as soon as they were synthesized, the ability of PCL5 to enhance DNR accumulation was retained, thus suggesting PCL200 as a promising delivery system for encapsulating P-gp inhibitors, such as PCL5.

Next-generation DNA sequencing of Panax samples revealed new genotypes: Burrows-Wheeler Aligner, Python-based abundance and clustering analysis.

Background: There are two major species of the Panax genus, namely Panax ginseng and Panax quinquefolius. Other than the nucleic acid test and nucleic acid amplification test, DNA sequencing can be used to authenticate the species of ginseng samples, especially when their physical forms cannot be used for differentiation. Method: In this work, next generation sequencing was used to obtain millions of reads from fourteen ginseng samples (root, powder, and granule). Then Gaussian Mixture clustering analysis was applied to analyze the reads from each sample. Results and Discussion: A new genotype has been revealed in this study. Two samples have been authenticated with certainty, while the others may be hybrid in nature as revealed by the clustering results.

Cytosolic Calcium Measurement Utilizing a Single-Cell Biochip to Study the Effect of Curcumin and Resveratrol on a Single Glioma Cell.

A microfluidic method has been developed for real-time measurement of the effects of curcumin on the intracellular calcium concentration in a single glioma cell (U87-MG). This method is based on quantitative fluorescence measurement of intracellular calcium in a cell selected in a single-cell biochip. This biochip consists of three reservoirs, three channels, and a V-shaped cell retention structure. Because of the adherent nature of glioma cells, a single cell can adhere within the aforementioned V-shaped structure. The single-cell calcium measurement will minimize cell damage caused by conventional cell calcium assay methods. Previous studies have shown that curcumin increased cytosolic calcium in glioma cells using the fluorescent dye: Fluo-4. So in this study, the effects of 5 µM and 10 µM solutions of curcumin on the increases of cytosolic calcium in a single glioma cell have been measured. Moreover, the effects of 100 µM and 200 µM of resveratrol are measured. At the final stage of the experiments, ionomycin was used to increase the intracellular calcium to the highest possible level due to dye saturation. It has been demonstrated that microfluidic cell calcium measurement is a real-time cytosolic assay that requires small quantities of reagent, which will have potential uses for drug discovery.

Development of organic three-phase laminar flow microfluidic chip for extraction of ginsenosides from Panax ginseng.

BACKGROUND: Herbal extracts contain multiple active constituents, so the sample preparation based on the liquid-liquid extraction (LLE) is demanding, especially when a study subsequent to extraction is needed. Since the laminar flow occurring in microchannels can be formed between two miscible organic phases, a new method of extracting polar compounds from the crude extract of Panax ginseng Meyer in aqueous ethanol by pure n-butanol in the three-phase laminar flow microfluidic chip was established. METHODS: A new chip consisting of long microchannels with a guide structure was employed to improve the extraction efficiency caused by the low diffusion ability of saponins. The method was evaluated by using the extraction yields and purities of ginsenosides Rg1, Re and Rb1 as the indicators, and extraction conditions such as flow rate, temperature and other governing factors were optimized. RESULTS: Using the new chip method, the extraction efficiencies of ginsenoside Rg1, Re and Rb1 were 63.1%, 69.5% and 71.6%, respectively, which are higher than the 26% achieved in a previous report. The extraction yields of 1.53, 0.51, 0.90 mg/g were also higher than those obtained previously by the successive laminar flow microchip method. CONCLUSION: The proposed new microfluidic chip method has simplified the sample pretreatment steps to improve the yield of ginsenoside extraction from ginseng samples.

Optimization of a microfluidic microarray device for the fast discrimination of fungal pathogenic DNA.

A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two poly(dimethylsiloxane) (PDMS)-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the preprinted radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between replicate samples. The effect of microchannel depth was studied, and hybridization time is predictable at a certain rotation speed. Cy5 dye labels were proved to show much higher hybridization efficiency as well as less photobleaching effect as compared with the fluorescein dye labels used in our previous work. With these optimized conditions, the method was applied to the detection of three fungal pathogenic polymerase chain reaction (PCR) products with a sample load of 0.2 ng (in 1 microl). Furthermore, the single-base-pair discrimination between the PCR products of two relevant Botrytis species (B. cinerea and B. squamosa) was achieved in a duration as short as 3 min.

Simultaneous determination of free and total paclitaxel in blood in a three-phase laminar flow microchip.

In this study, a three-phase laminar flow microfluidic chip (TPL chip) combined with HPLC was developed for monitoring free and total concentrations of paclitaxel (PTX) in blood simultaneously. A diluted whole blood sample (aqueous phase) was introduced into the chip, ethyl acetate (organic phase) was introduced into the chip for extraction, and an interphase was used to prevent the blood sample from coming into direct contact with the organic phase. Because only free drug can quantitatively diffuse into the organic extraction phase and the free drug fraction has a linear relationship with the dilution factor of blood, both the free and total drug concentrations can be obtained by detecting the concentration of paclitaxel in the organic extraction phase. The governing factor such as flow rate for extraction was optimized. Docetaxel was used as an internal standard. The reliability of the quantitative diffusion of molecules in the TPL chip was proved by the methodological investigation of PTX in PBS sample, which showed a good linearity in the concentration range of 0.5 - 100 µg/mL and a detection limit of 7 ng/mL. Good repeatibilities for retention time (RSD of PTX is 1.23%, docetaxel is 1.14%, n = 5) and peak area ratio of PTX to docetaxel (RSD is 4.38%) were obtained. For blood sample analysis, only 100 µL of sample was needed and whole pretreatment was finished in 35 min, and a recovery of 94~117% were obtained. The provided method showed advantages in fast analysis speed, minimum sample handing, and potential ability of automation, and integration.

Construction of an Array of Photonic Crystal Films for Visual Differentiation of Water/Ethanol Mixtures.

A photonic crystal film (PCF) which consists of a porous layered structure with a highly ordered periodic arrangement of nanopores has been used to differentiate between various mixtures of water and ethanol (EtOH). The refractive index difference between the wall (silica) of the empty nanopore and air which occupies it results in the structural color of the PCF. This color disappears when the nanopores are infiltrated by a liquid with a similar refractive index to silica (or silicon dioxide). The disappearance of the structural color provides a means to construct a colorimetric sensor to differentiate between various water/EtOH mixtures based on their wettability of the nanopores in the PCF. In this study, an array of silica-based PCFs was synthesized on a silicon substrate with a precise control of nanopore properties using the co-assembly/sedimentation method. Using this method, we benefitted from having different PCFs on a single substrate. Chemical coatings, neck angles, and film thicknesses on each PCF were the three factors used to adjust the wettability of the pores. Nanopore wetting by water/EtOH mixtures was studied in a systematic manner based on the three factors, and the findings were used to develop a sensor for visual differentiation of various water/EtOH mixtures. The final developed sensor consisting of an array of six PCFs was able to differentiate between seven different water/EtOH mixtures: W10, W20, W30, W40, W50, W60, and W70, in which W10 means 10% of water in EtOH.

The microfluidic capture of single breast cancer cells for multi-drug resistance assays.

Utilizing the microfluidic single-cell technique enables us to study the inhibition of multidrug resistance due to drug efflux on a single triple-negative breast cancer cell. This method examines drug efflux inhibition on a single cell in a microfluidic chip using a conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, the integration of single-cell selection, dye and drug loading, and fluorescence measurement for intracellular drug accumulation is all conducted on a single microfluidic chip. By using a microfluidic chip and the adherent nature of the cancer cell lines, a single breast cancer cell could be selected and retained near the cell retention structure in the chip. This enabled us to detect dye accumulation in the MDR breast cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 (Rh123) were used as the P-glycoprotein (P-gp) inhibitor and fluorescent dye, respectively. Furthermore, Paclitaxel, a commonly known chemotherapeutic used in breast cancer patients, was administered in the presence of both reagents. During the entirety of the experiment fluorescence measurement was used to monitor the fluctuating levels of intracellular Rh123 levels, and an optical imaging system was used to monitor the shape and size of the cell. The results showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control due to the competitive inhibition of paclitaxel and the non-competitive inhibition subjected by CsA.

Nucleic acid microarrays created in the double-spiral format on a circular microfluidic disk.

A microfluidic microarray that is created in the double spiral format has produced a greater density of probes than in our previous report. Using this double-spiral format together with centrifugal pumping for liquid delivery, 384 x 384 hybridization assays have been performed on one circular disk at one time, at the intersections between the spiral channels and spiral probe lines. Each sample was introduced into each inlet reservoir leading to 4 spiral channels and was analyzed independently, and so the hybridization results were self-corrected among the 4 spiral channels. In this work, fast microarray hybridizations have been successfully achieved by using both complementary oligonucleotides as well as PCR products prepared from plant fungal pathogen cultures.

Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip.

Since multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy, we report a microfluidic approach combined with the same-single-cell analysis to investigate the modulation of MDR, manifested as the inhibition of drug efflux. A microfluidic chip that was capable of selecting and retaining a single multidrug-resistant cancer cell was used to investigate drug efflux inhibition in leukemia cell lines. Three advantages of the microfluidic-based same-single-cell analysis (dubbed as SASCA) method have been revealed. First, it readily detects the modulation of drug efflux of anticancer compounds (e.g., daunorubicin) by MDR modulators (e.g., verapamil) among cellular variations. Second, SASCA is able to compare the different cellular abilities in response to drug efflux modulation based on the drug transport kinetics of single cells. Third, SASCA requires only a small number of cells, which may be beneficial for investigating drug resistance in minor cell subpopulations (e.g., cancer "stem" cells).

Inkjet printed electrode arrays for potential modulation of DNA self-assembled monolayers on gold.

In this paper, we report a novel and cost-effective fabrication technique to produce electrode arrays that can be used for monitoring and electrical manipulation of the molecular orientation of DNA self-assembled monolayers (SAMs) on gold. The electrode arrays were prepared from gold coated glass sides or compact discs (CD-Rs) by using standard office inkjet printers without any hardware or software modifications. In this method, electrode arrays of varied shape and size (from submillimeter to centimeter) can be rapidly fabricated and are suitable for standard electrochemical measurements. We were able to use a dual-channel potentiostat to control the electrodes individually and a fluorescence (FL) scanner to image the electrode array simultaneously. With such an integrated modulation setup, the structural switching behavior (from "lying" to "standing" position) and the enhanced hybridization reactivity of thiolate DNA SAMs on gold under potential control have been successfully demonstrated.

From traditional Chinese medicine to rational cancer therapy.

Many natural products and derivatives thereof belong to the standard repertoire of cancer chemotherapy. Examples are Vinca alkaloids, taxanes and camptothecins. In recent years, the potential of natural products from plants, notably from medicinal plants used in traditional Chinese medicine (TCM), has been recognized by the scientific community in the Western world. To provide an example of the most recent developments in this field, we have selected several compounds, namely artesunate, homoharringtonine, arsenic trioxide and cantharidin, that are found in natural TCM products and that have the potential for use in cancer therapy. Controlled clinical studies have shown that homoharringtonine and arsenic trioxide can exert profound activity against leukaemia. Increased knowledge of the molecular mechanisms of TCM-derived drugs and recent developments in their applications demonstrate that the combination of TCM with modern cutting-edge technologies provides an attractive strategy for the development of novel and improved cancer therapeutics.