Design and development of ferrocene-containing chitosan-cografted-branched polyethylenimine redox conjugates for monitoring free flap failure after reconstructive surgery.

We reported a proof-of-concept study of developing an electrochemical biosensor for prolonged continuous monitoring free flap failure caused by vascular occlusion after reconstructive surgery. Ferrocene (Fc)-containing Chitosan-cografted-Branched Polyethylenimine redox conjugates (CHIT-Fc-co-BPEI-Fc) were used as pH-tuneable matrix to attach the target enzymes (glucose oxidase ≡ GOD and lactate oxidase ≡ LOD, respectively) to build up the corresponding GOD-sensor and LOD-sensor. The sensitivity of GOD-/LOD-sensor was found to be 2.89(±0.06)µA/mM and 2.95(±0.19)µA/mM with a limit of detection (LOD) of 0.047 mM and 0.172 mM, respectively. The sensor performance was further pre-clinically validated via rabbit study, which confirmed that the designed biosensors could electrochemically detect the changes of metabolite levels caused by flap failure within minutes. This sensor protocol is able to be fabricated as embedded biosensor for prolong continuous detection for clinical application/research.

Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. METHODS: Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA)® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. RESULTS: We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. CONCLUSIONS: Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses.

Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

BACKGROUND AND OBJECTIVES: Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. METHODS: In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. RESULTS: The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. CONCLUSION: Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells.

Herein we present a lab-chip device for highly efficient and rapid detection of circulating tumor cells (CTCs) from whole blood samples. The device utilizes a microfabricated silicon microsieve with a densely packed pore array (10(5) pores per device) to rapidly separate tumor cells from whole blood, utilizing the size and deformability differences between the CTCs and normal blood cells. The whole process, including tumor cell capture, antibody staining, removal of unwanted contaminants and immunofluorescence imaging, was performed directly on the microsieve within an integrated microfluidic unit, interconnected to a peristaltic pump for fluid regulation and a fluorescence microscope for cell counting. The latter was equipped with a dedicated digital image processing program which was developed to automatically categorize the captured cells based on the immunofluorescence images. A high recovery rate of >80% was achieved with defined numbers of MCF-7 and HepG2 cancer cells spiked into human whole blood and filtered at a rapid flow rate of 1 mL min(-1). The device was further validated with blood drawn from various cancer patients (8 samples). The whole process, from sample input to result, was completed in 1.5 h. In addition, we have also successfully demonstrated on-microsieve fluorescence in situ hybridization for single cell molecular analysis. This simple method has great potential to supplant existing complex CTC detection schemes for cancer metastasis analysis.

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis.

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

TopDown real-time gene synthesis.

This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.

De novo gene synthesis design using TmPrime software.

This chapter presents TmPrime, a computer program to design oligonucleotide for both ligase chain reaction (LCR)- and polymerase chain reaction (PCR)-based de novo gene synthesis. The program divides a long input DNA sequence based on user-specified melting temperatures and assembly conditions, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences, and potential formation of secondary structures in a PDF file, which will be sent to the user via e-mail. The program also provides functions on sequence pooling to separate long genes into smaller pieces for multipool assembly and codon optimization for expression based on the highest organism-specific codon frequency. This software has been successfully used in the design and synthesis of various genes with total length >20 kbp. This program is freely available at http://prime.ibn.a-star.edu.sg.

New insights into the de novo gene synthesis using the automatic kinetics switch approach.

Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6kbp from 1nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74kbp from 1nM oligonucleotide.

Hybrid sample-inverted reflow and soft-lithography technique for fabrication of conicoid microlens arrays.

We report a cost-effective fabrication method, with a combination of the sample-inverted reflow technique and the soft-lithography replication method, to fabricate conicoid refractive microlens arrays (MLAs), including hyperboloid, paraboloid, and ellipsoid MLAs in inorganic-organic hybrid SiO2-ZrO2 solgel material. The fabrication procedures involve two basic steps. First, a master of the conicoid MLA was made in photoresist by the sample-inverted reflow technique. Second, we built a negative mold of the master by casting polydimethylsiloxane (PDMS) onto a silicone elastomer against the master, and then the profile was imprinted onto the solgel glass. As a result, the fabricated solgel MLAs have been obtained with excellent smooth profiles, having negligible discrepancies from the profiles of ideal conicoid MLAs.

Reflowed solgel spherical microlens for high-efficiency optical coupling between a laser diode and a single-mode fiber.

To improve the coupling efficiency between a laser diode and a single-mode fiber, we propose a two-microlens coupling scheme that uses two solgel spherical microlenses for high coupling efficiency. The conventional reflow technique was employed and extended to the inorganic-organic hybrid SiO2/ZrO2 solgel material to form the microlenses. Preliminary results show that the coupling efficiency was increased to--1.28 dB (74.5%) by the proposed scheme, compared with a coupling efficiency of--10.13 dB (9.7%) by the butt-joint method. The proposed fabrication technique demonstrates that use of a reflowed solgel spherical microlens is a cost-effective mass-production approach to application of micro-optical elements in optical communication.

Fabrication of concave refractive microlens arrays in solgel glass by a simple proximity-effect-assisted reflow technique.

We report a simple method for fabricating a concave refractive microlens array (MLA) in solgel glass by using a proximity-effect-assisted reflow technique. The solgel concave refractive MLA that we fabricated had excellent surface smoothness; good dimensional conformity, with an 8.23% nonuniformity of the microlens elements; and structural perfection, with a biggest deviation of 1% from a perfect concave spherical crown. The relative error between the measured and the designed values of the concave MLA's focal length was only 1.83%. Compared with the conventional fabrication techniques for concave MLAs, the proposed method has significant advantages including simplicity, low cost, good element conformity, and smooth device surface.