This critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration, automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094–1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology. This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes. In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into technical advances, major market segments and promising applications. We continue with a detailed characterization of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations, application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for microfluidic platforms which is based on their characteristics according to key requirements of different applications and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit operations and the flexibility in programming different liquid handling protocols (295 references).

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Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications

The Kinase Akt1 Controls Macrophage Response to Lipopolysaccharide by Regulating MicroRNAs

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

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Fibroblast-specific TGF-β–Smad2/3 signaling underlies cardiac fibrosis

The application of dielectrophoretic field-flow fractionation (depFFF) to the isolation of circulating tumor cells (CTCs) from clinical blood specimens was studied using simulated cell mixtures of three different cultured tumor cell types with peripheral blood. The depFFF method can not only exploit intrinsic tumor cell properties so that labeling is unnecessary but can also deliver unmodified, viable tumor cells for culture and/or all types of molecular analysis. We investigated tumor cell recovery efficiency as a function of cell loading for a 25 mm wide x 300 mm long depFFF chamber. More than 90% of tumor cells were recovered for small samples but a larger chamber will be required if similarly high recovery efficiencies are to be realized for 10 mL blood specimens used CTC analysis in clinics. We show that the factor limiting isolation efficiency is cell-cell dielectric interactions and that isolation protocols should be completed within approximately 15 min in order to avoid changes in cell dielectric properties associated with ion leakage.

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Isolation of rare cells from cell mixtures by dielectrophoresis

Lateral flow assays (LFAs) have attracted interest due to their friendly user formats, short assay times, little interferences, low costs, and being easy by operated by non-specialized personnel. This technique is based on biochemical interaction of antigen-antibody or probe DNA-target DNA hybridization. A lateral flow assay (LFA) is composed of four parts: a sample pad, which is the area on which sample is dropped; conjugate pad, on which labeled tags combined with biorecognition elements; reaction membrane containing test line and control line for target DNA-probe DNA hybridization or antigen-antibody interaction; and absorbent pad, which reserves waste. For the construction of LFAs gold nanoparticles, colored latex beads, carbon nanoparticles, quantum dots, and enzymes are used as a label for increasing the sensitivity. In this work, the principle of LFAs, biorecognition elements, analytical performances, limits of detection (LODs), linear ranges of developed LFAs in different fields are summarized. Future perspectives in this area are also discussed.

Lateral flow assays: Principles, designs and labels

Replacing antibodies with aptamers in lateral flow immunoassay.

Gold nanoparticle (AuNP) based colorimetric aptasensor have been developed for many analytes recently largely because of the ease of detection, high sensitivity, and potential for high-throughput analysis. Most of the target aptamers for detection have short sequences. However, the approach shows poor performance in terms of detection sensitivity for most of the long-sequence aptamers. To address this problem, for the first time, we split the 76 mer aptamer of 17β-estradiol into two short pieces to improve the AuNP based colorimetric sensitivity. Our results showed that the split P1 + P2 still retained the original 76 mer aptamer's affinity and specificity but increased the detection limit by 10-fold, demonstrating that as low as 0.1 ng/mL 17β-estradiol could be detected. The increased sensitivity may be caused by lower aptamer adsorption concentration and a lower affinity to the AuNPs of a short single-strand DNA (ssDNA) sequence. Our study provided a new way to use long-sequence aptamers to develop a highly sensitive AuNP-based colorimetric aptasensor.

/pdf/highly-sensitive-colorimetric-detection-of-17b-estradiol-3ps1j942ce.pdf

Highly sensitive colorimetric detection of 17β-estradiol using split DNA aptamers immobilized on unmodified gold nanoparticles

Recent developments in application of stable isotope analysis on agro-product authenticity and traceability.

Fast immunoassay-based screening methods are unavailable for most small-molecule pesticides because of a lack of immunogenicity and the difficulty in obtaining antibodies by animal immunization. Aptamers are single-stranded DNA molecules selected through an in vitro process, which can bind to any target including nonimmunogenic small molecules with high affinity and specificity. Although various aptamer-based sensing methods have been developed for antibiotics, microorganisms, heavy metal ions, and biotoxins, there are few reports on aptamer-based methods for quick detection of organophosphorous pesticides. The gold (Au) nanoparticle (AuNP) colorimetric assay is a widely utilized rapid detection method because of properties such as easy operation and visualized results. In the present study, organophosphorous pesticide aptamers were adsorbed on the surface of AuNPs to stabilize the AuNP solution against high concentrations of salt to prevent AuNP aggregation. After the addition of targets, the aptamers binding to the targets are detached from the AuNPs, resulting in aggregation of AuNPs and a color change from red to purple-blue. The proposed method can detect 6 organophosphorous pesticides with good recoveries from 72% to 135% in environmental river water samples. The present study provides a new way for simple, rapid, and multiplex detection of organophosphorous pesticides.

Gold nanoparticle–based colorimetric aptasensor for rapid detection of six organophosphorous pesticides

Most aptasensors are designed based on aptamers' conformational change or structural transformation upon interaction with their targets. A limitation of these strategies is the unpredictability of such structural alterations that may yield false-positive or nonspecific signals. In bioanalysis, a dominant strategy for protein determination is the sandwich assay because of its high specificity and sensitivity resulting from the dual recognition mechanism. However, the approach is poorly suited to detection of low molecular weight targets, which unlikely bind to two aptamers simultaneously due to steric hindrance. To circumvent this drawback, a split aptamer strategy was recently developed, in which nucleic acid aptamers were split into two fragments that could specifically form a ternary assembly in the presence of ligand. This elegant approach has been extensively adopted for detection of various targets, especially small molecular weight targets, with different transduction methods including colorimetric, fluorescence, and electrochemical techniques. This review first introduced split aptamers and how to obtain split aptamers, and then summarized recent advances in the development of sandwich-format biosensing assays based on split aptamer fragments, aiming at providing a general guide for the design of split-aptamer-based sandwich assays. Furthermore, we discussed possible challenges in the development of split-aptamer-based sandwich assay regarding stability, sensitivity and multiplicity, so as to offer future opportunities of this assay.

Ailiang Chen

Papers

Replacing antibodies with aptamers in lateral flow immunoassay.

Highly sensitive colorimetric detection of 17β-estradiol using split DNA aptamers immobilized on unmodified gold nanoparticles

Recent developments in application of stable isotope analysis on agro-product authenticity and traceability.

Gold nanoparticle–based colorimetric aptasensor for rapid detection of six organophosphorous pesticides

Split aptamers and their applications in sandwich aptasensors