Chiuan-Chian Chiou

Background/Objectives: The PowerMag system (PM) is a platform for the isolation of circulating tumor cells (CTCs) by the depletion of CD45⁺-leukocytes. However, an EpCAM⁻CD45⁻ cell population is present in large numbers in the cell filtrates collected by PM. This lowers the purity of the CTCs and negatively impacts their molecular characterization. The aims of this study are to characterize the cellular properties of the EpCAM⁻CD45⁻ cells and to upgrade the system to improve CTC purity. Methods: A real-time RT-PCR assay, Liu’s stain analysis, and Annexin V (AnxV) binding assay were used to define the cellular properties of the EpCAM⁻CD45⁻ cells. An upgraded system was developed to remove the EpCAM⁻CD45⁻ cells and improve the CTC purity. Clinical blood samples were used to evaluate the performance of the system. Results: The EpCAM⁻CD45⁻ cells were defined as apoptotic cells, which displayed apoptotic body-like morphology and elicited AnxV binding activity. AnxV beads developed in-house can effectively bind and remove EpCAM⁻CD45⁻ cells from the cell filtrates. An improved generation of a CTCs isolation platform, designated as PM II, was developed by integration of AnxV beads into the workflow to remove the apoptotic cells. PM II recovered CTCs with improved CTC purity by effective removal of the background apoptotic cells. The improved performance of PM II allowed for direct profiling of cancer-related gene mutations by next-generation sequencing without cell picking and further purification. Conclusions: PM II holds great promise as a platform for isolating CTCs with improved purity and for exploring its application in cancer diagnosis and monitoring in a clinical setting.

This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating. Additionally, using two fluorescence intensity thresholds enabled simultaneous detection and counting of positive and negative droplets within a single fluorescence detection channel. The chip can partition approximately 6000 droplets from a 5 µL sample within 10 min, with a droplet diameter of around 110 µm and a coefficient of variation (CV) value of 0.82%. Staphylococcus aureus was quantified via the proposed platform. The results demonstrated a highly accurate correlation coefficient (R = 0.9998), and the detection limit reached a concentration of 1.7 × 10² copies/µL. The entire process of the droplet digital LAMP reaction, from droplet generation to incubation to quantitative results, took a maximum of 70 min.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads). The results showed that in a cultured medium, the majority of mRNA and lncRNA exist in larger EVs, whereas miRNA exist in both large and small EVs from the differential centrifugation fractions. All these RNA species exist in ANX beads captured EVs. We then used ANX beads to capture EVs in plasma samples from non-small-cell lung cancer patients and age-matched healthy volunteers. We found that the ANX bead capturing could efficiently improve RNA detection from human plasma, compared with direct extraction of RNA from plasma. Using ANX-bead capturing and reverse transcription and quantitative PCR, we detected significantly higher levels of CK19 mRNA, MALAT1 lncRNA, and miR155 miRNA in the plasma of lung cancer patients. These facts suggested the collection methods strongly affect the results of exRNA measurement from EVs, and that ANX beads can be a useful tool for detecting exRNA from plasma samples in clinical application.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30–40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.

Background/Aim: Circulating mRNA can be a useful source of cancer biomarkers. We took advantage of direct transcriptomic analysis in plasma RNA to identify novel mRNA markers for non-small cell lung cancer (NSCLC). Patients and Methods: Plasma RNA from NSCLC patients and healthy individuals was profiled with cDNA-mediated annealing, selection, extension and ligation (DASL) microarrays. The microarray results were further validated in plasma RNA. Results: Through RNA profiling and online database mining, four gene transcripts were filtered as candidate markers of NSCLC. After validation, the PCTAIRE-1 transcript was identified as a circulating mRNA marker. The diagnostic potential of PCTAIRE-1 was evaluated by receiver operating characteristic curve analysis, which gave a sensitivity and specificity of 60% and 85%, respectively. In addition, high plasma PCTK1 levels were also correlated with poor progression-free survival (p=0.008). Conclusion: Circulating mRNA can be profiled with the DASL assay. From the profile, PCTAIRE-1 RNA in the plasma we discovered as a novel diagnostic/prognostic biomarker and an indicator of poor survival in NSCLC patients.

The nanopattern of highly ordered and uniform Au nanoporous membranes with different sizes and thicknesses and the downscaling approach through the combination of colloidal based nanosphere lithography (NSL) and thermal evaporation was proposed to fabricate an extending-gate field effect transistor (EGFET) membrane. The fabrication involved the use of PS nanospheres templates of 500 nm and 100 nm in diameters and various Au thickness of 10, 25 and 40 nm. Carried out in the detection of Staphylococcus aureus 16S rRNA hybridization test with analytical range of 10¹–10⁶ pM DNA targets, the smaller the Au nanoporous diameter made up by the thicker Au layer produced a gradual improvement in potentiometric study. The Au-nanoporous produced by the thickest Au film at 40 nm and smaller diameter of PS nanospheres (100 nm) demonstrated the most optimum threshold voltage shift and limit of detection (LOD) of ∼1 pM altogether with remarkable specificity in the presence of highly concentrated non-specific DNA of other pathogens. Analytical outcomes point out that smaller, periodic and uniform nanoporous EGFET membrane facilitated the larger hybridization signal due to the higher active surface area enabling the more optimum control of DNA orientation and immobilization.

McCune-Albright syndrome (MAS) is characterized by the triad of precocious puberty, café au lait pigmentation, and polyostotic fibrous dysplasia (FD) of bone, and is caused by post-zygotic somatic mutations—R201H or R201C—in the guanine nucleotide binding protein, alpha stimulating (GNAS) gene. In the present study, a novel peptide nucleic acid (PNA) probe with fluorescent labeling was designed to detect trace amounts of somatic mutant GNAS in a single tube reaction. The method was applied to screen GNAS mutations in six patients with MAS/FD. The results showed that the PNA probe assay could detect low abundant mutants in 200-fold excess of wild-type alleles. The GNAS mutation was found in three patients with severe disease (MAS) by using the assay. The other three patients with mild disease (having only FD) showed a wild-type result. This study has provided a simple method to detect trace amounts of GNAS mutants with high sensitivity in large amounts of wild-type DNA.

Zinc oxide nanograss (ZnONG) decorated with gold (Au) nanospeckles is demonstrated as a sensing platform for bacterial DNA hybridization in this study. Thermal evaporation of 5 nm Au on hydrothermally synthesized ZnONG generates highly dense speckles, which contributes to an approximately two-fold enhancement of the effective surface area and diffusion coefficient in cyclic voltammetry (CV) measurement compared to the non-speckled nanograss. Selective capture of molecular probes from our predesigned sequence of S. epidermidis 16S rRNA onto nanospeckled ZnONG indicated a specific interaction with the DNA target from this prevalently isolated skin pathogen through hybridization screening. The fabricated DNA biosensor demonstrated great ability to quantify a wide range of complementary DNA target, from 10 pM to 1 μM. Moreover, it attained an impressive limit of detection (LOD) of 0.506 pM, as determined by the charge-transfer resistance (R_CT), upon DNA hybridization in an electrochemical impedance spectroscopy (EIS) analysis. The proposed structure of the hybrid Au-speckled ZnONG effectively improved the electro-catalytic properties and electron migration of electrochemical DNA detection due to the increment of the surface area, which consequently renders this structure a potential platform for array-based DNA sensor development.

Background: Mutation of epidermal growth factor receptor (Methods: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types ofResults: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the threeConclusions: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of