Microfluidic device capable of sensing ultrafast chemiluminescence

Based on the principle of liquid core waveguide, a novel microfluidic device with micro-scale detection window capable of sensing flashlight emitted from rapid 1,1′-oxalyldi-4-methylimidazole (OD4MI) chemiluminescence (CL) reaction was fabricated. Light emitted from OD4MI CL reaction occurring in the micro-dimensional pentagonal detection window (length of each line segment: 900.0 μm, depth: 50.0 μm) of the microfluidic device with two inlets and one outlet was so bright that it was possible to take an image every 1/30 s at the optimal focusing distance (60 cm) using a commercial digital camera. Peaks obtained using a flow injection analysis (FIA) system with the micro-scale detection window and OD4MI CL detection show excellent resolution and reproducibility without any band-broadening observed in analytical devices having additional reaction channel(s) to measure light generated from slow CL reaction. Maximum height (H_max) and area (A) of peak, reproducibility and sensitivity observed in the FIA system with the microfluidic device and OD4MI CL detection depends on (1) the mole ratio between bis(2,4,6-trichlorophenyl) oxalate and 4-methyl imidazole yielding OD4MI, (2) the flow rate to mix OD4MI, H₂O₂ and 1-AP in the detection window of the microfluidic device, and (3) H₂O₂ concentration. We obtained linear calibration curves with wide dynamic ranges using H_max and A. The detection limit of 1-AP determined with H_max and A was as low as 0.05 fmole/injection (signal/background = 3.0).     

A hydrogel-based microfluidic device for the studies of directed cell migration

We have developed a hydrogel-based microfluidic device that is capable of generating a steady and long term linear chemical concentration gradient with no through flow in a microfluidic channel. Using this device, we successfully monitored the chemotactic responses of wildtype Escherichia coli (suspension cells) to alpha-methyl-DL-aspartate (attractant) and differentiated HL-60 cells (a human neutrophil-like cell line that is adherent) to formyl-Met-Leu-Phe (f-MLP, attractant). This device advances the current state of the art in microchemotaxis devices in that (1) it demonstrates the validity of using hydrogels as the building material for a microchemotaxis device; (2) it demonstrates the potential of the hydrogel based microfluidic device in biological experiments since most of the proteins and nutrients essential for cell survival are readily diffusible in hydrogel; (3) it is capable of applying chemical stimuli independently of mechanical stimuli; (4) it is straightforward to make, and requires very basic tools that are commonly available in biological labs. This device will also be useful in controlling the chemical and mechanical environment during the formation of tissue engineered constructs.     

Quantification of taurine and amino acids in mice single fibrosarcoma cell by microchip electrophoresis coupled with chemiluminescence detection

A method based on MCE coupled with chemiluminescence (CL) detection was developed for the determination of taurine (Tau) and amino acids including alanine (Ala), glycine (Gly), tryptophan (Trp), glutamic acid (Glu) and aspartic acid (Asp) present in mice single fibrosarcoma (S180) cells. Cell injection, loading, cytolysis, electrophoretic separation and CL detection were integrated onto a simple double‐T microfluidic chip. The intracellular constituents were electrophoretically separated within 150 s. The CL detection was based on the enhancement effects of Tau and amino acids on the CL reaction of luminol with H₂O₂ and Cu²⁺. The average amounts of Tau, Trp, Gly, Ala, Glu and Asp in per S180 cell from a cell population were 4.73, 1.23, 2.65, 1.94, 1.61 and 1.99 fmol. Ten S180 cells were analyzed, and the contents of Tau, Trp, Gly, Ala, Glu and Asp in mice single S180 cells were found to be in the range of 1.78–8.84, 0.95–2.31, 1.08–6.87, 1.03–4.05, 0.84–2.61 and 0.82–3.68 fmol, respectively. This work demonstrates that MCE coupled with CL detection is a useful analytical tool that is simple, quick and highly sensitive for single‐cell analysis.     

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3σ).     

Detection method for microchip separations

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.     

Microchip electrophoresis with chemiluminescence detection for assaying ascorbic acid and amino acids in single cells

A method based on microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed for the determination of ascorbic acid (AA) and amino acids including tryptophan (Trp), glycine (Gly) and alanine (Ala) present in single cells. Cell injection, loading, lysing, electrophoretic separation and CL detection were integrated onto a simple cross microfluidic chip. A single cell was loaded in the cross intersection by electrophoretic means through applying a set of potentials at the reservoirs. The docked cell was lysed rapidly under a direct electric field. The intracellular contents were MCE separated within 130 s. CL detection was based on the enhancing effects of AA and amino acids on the CL reaction of luminol with K₃[Fe(CN)₆]. Rat hepatocytes were prepared and analyzed as the test cellular model. The average intracellular contents of AA, Trp, Gly and Ala in single rat hepatocytes were found to be 38.3, 5.15, 3.78 and 3.84 fmol (n = 12), respectively.     

The Enhancing Immune Response and Anti-Inflammatory Effects of Caulerpa lentillifera Extract in RAW 264.7 Cells

Background: Caulerpa lentillifera (CL) is a green seaweed, and its edible part represents added value as a functional ingredient. CL was dried and extracted for the determination of its active compounds and the evaluation of its biological activities. The major constituents of CL extract (CLE), including tannic acid, catechin, rutin, and isoquercetin, exhibited beneficial effects, such as antioxidant activity, anti-diabetic activity, immunomodulatory effects, and anti-cancer activities in in vitro and in vivo models. Whether CLE has an anti-inflammatory effect and immune response remains unclear. Methods: This study examined the effect of CLE on the inflammatory status and immune response of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the mechanisms involved therein. RAW264.7 cells were treated with different concentrations of CLE (0.1–1000 µg/mL) with or without LPS (1 µg/mL) for 24 h. Expression and production of the inflammatory cytokines, enzymes, and mediators were evaluated. Results: CLE suppressed expression and production of the pro-inflammatory cytokines IL-6 and TNF-α. Moreover, CLE inhibited expression and secretion of the inflammatory enzyme COX-2 and the mediators PGE2 and NO. CLE also reduced DNA damage. Furthermore, CLE stimulated the immune response by modulating the cell cycle regulators p27, p53, cyclin D2, and cyclin E2. Conclusions: CLE inhibits inflammatory responses in LPS-activated macrophages by downregulating inflammatory cytokines and mediators. Furthermore, CLE has an immunomodulatory effect by modulating cell cycle regulators.     

Chemiluminescence of Peracetic Acid in Alkaline Medium and its Application to Dihydralazine Sulfate Determination

The chemiluminescence (CL) of peracetic acid (PAA) in alkaline medium is very weak but is strongly enhanced after the addition of dihydralazine sulfate (DHZS). Based on this phenomenon, a simple, rapid and highly sensitive flow-injection CL method for the determination of DHZS was developed. The CL emission was linearly related to the DHZS concentration in the range of 20–4000 ng mL⁻¹ with a detection limit (3σ) of 1.2 ng mL⁻¹. As a preliminary application, the proposed method was successfully applied to the determination of DHZS in pharmaceutical preparations; the recovery of DHZS in human urine was between 96.5% and 102.2%. A detailed CL mechanism was proposed and singlet molecular oxygen (¹O₂) was suggested to be produced in the CL reaction process.     

A Biphasic Mercury‐Ion Sensor: Exploiting Microfluidics to Make Simple Anilines Competitive Ligands

Combining the molecular wire effect with a biphasic sensing approach (analyte in water, sensor‐dye in 2‐methyltetrahydrofuran) and a microfluidic flow setup leads to the construction of a mercury‐sensitive module. We so instantaneously detect Hg²⁺ ions in water at a 500 μM concentration. The sensor, conjugated non‐water soluble polymer 1 (XFPF), merely supports dibutylaniline substituents as binding units. Yet, selective and sensitive detection of Hg²⁺‐ions is achieved in water. The enhancement in sensory response, when comparing the reference compound 2 to that of 1 in a biphasic system in a microfluidic chip is >10³. By manipulation of the structure of 1 , further powerful sensor systems should be easily achieved.     

Lab-on-paper-based devices using chemiluminescence and electrogenerated chemiluminescence detection

As an analytical support, paper, being low cost, highly abundant, of high porosity, disposable or biodegradable, and easy to use, store, transport, and print, has excellent chemical compatibility with many applications. Since the first microfluidic paper-based analytical device (μ-PAD or lab-on-paper) was proposed, the paper-based assay has never attracted as much attention as it does now. There has recently been rapidly increasing interest in using sensitive luminescence methods, for example chemiluminescence (CL) and electrogenerated chemiluminescence (ECL), as the detection strategy for lab-on-paper devices. Because of their intrinsic characteristics, CL and ECL provide outstanding performance while retaining the simplicity, low cost, multifunctionality, versatility, flexibility, and disposability of μ-PADs. The objective of this review is to cover the development of lab-on-paper-based devices using CL and ECL detection, including fabrication of paper devices, construction of sensing interfaces, signal amplification strategies, external instruments used, and applications. We believe that lab-on-paper devices with CL and ECL detection methods will meet the diverse requirements of point-of-care diagnosis.     

3D Origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.     

Time-resolved chemiluminescence technique for the microdetermination of proteins based on their complexation with copper(II).

Based on the complexation between proteins and Cu(II) coupled with the time-resolved chemiluminescence (CL) technique, a highly sensitive and quantitative assay for measuring proteins in solution is described. The complexes of proteins with Cu(II) have a strongly catalytic effect on the luminol-H2O2 CL reaction. Because the CL emission produced by the complexes is much more long-lived than that by Cu(II), the CL signals originating from proteins can be easily identified and measured with a time-resolved technique. On this basis, bovine albumin fraction V (BAF V) can be quantitatively determined in the range of 0.01 - 5.0 microg/ml with a detection limit of 5.8 ng/ml. The results show that the proposed assay exhibits a small variation in the response values for the same amount of different proteins, as compared to the Lowry as well as Bradford assays. The CL assay has also been studied for the detection of immobilized proteins.     

A high throughput chemiluminescence method for determination of chemical oxygen demand in waters

A novel and high throughput chemiluminescence (CL) method for determination of chemical oxygen demand (COD) in water sample was originally developed based on potassium permanganate-glutaraldehyde CL system. With this method, dissolved organic matter in water samples was digested by excess acid potassium permanganate, the reacted mixture solutions containing surplus KMnO₄ were added in wells of a 96-well plate, followed by injection of glutaraldehyde in the wells, and CL was then produced along with the reaction of the added glutaraldehyde with the surplus KMnO₄ and detected by a photomultiplier tube (PMT). The difference (ΔI) between the CL intensity for distilled water and that for sample water was proportional to the COD value of water sample. The calibration graph was linear in the range of 0.16-19.24 mg L⁻¹ with a detection limit of 0.1 mg L⁻¹. A complete analysis could be performed in 40 min including digestion and detection, giving a very high throughput of 3 × 96 samples in about 60 min. Compared with the conventional methods, this method is simple and sensitive and consumes very limited and cheap reagents. Owing to its rapid, automatic, high throughput and low cost characteristics, the presented CL method has been applied successfully to the determination of COD in real water samples (n = 32) with satisfactory results.     

An on-column fracture/end-column reaction interface for chemiluminescence detection in capillary electrophoresis

Chemiluminescence (CL) offers a sensitive detection method for capillary electrophoresis (CE), but the implementation of CE–CL is usually under compromised operating conditions for CE, such as the prerequisite of extreme pH buffer for optimal CL reaction at the capillary outlet. This has sometimes significantly deteriorated the separation of CE. In this study, the development of a new interface makes it possible to optimize the operating conditions for CE separation and CL detection independently. The interface consists of an on-column fracture being installed in a reservoir near the capillary end to create an electrical connection and also serve as reagent addition entrance. The capillary terminal is inserted into an end-column reservoir for CL reaction and detection. In this arrangement, the applied electric field has been decoupled from the CL detection, which is proved to effectively improve CE's performance by allowing the use of optimal CE buffers. At the same time, it enables the optimization of CL detection independently. The applicability of this interface was evaluated by using acridinium ester (AE) and luminol systems. For AE system, the interfering products of CL reagent (⁻OH, HO₂⁻) have been prevented, and the pH range of CE buffer can be independent to the optimal pH value of AE CL reaction, which is usually below 3. The AE was detected using running buffer at pH 8.7, giving a detection limit of 0.1 nM (S/N = 3), and the theoretical plate numbers is as high as 56 000. The on-column fracture based configuration is simple, sensitive and easy to implement.     

A novel chemiluminescence technique for quantitative measurement of low concentration human serum albumin.

An efficient and highly sensitive chemiluminescence (CL) technique is proposed in the current study for detection of low levels of human serum albumin (HSA). Chemiluminescence (CL) produced during interaction between fluoresceinyl cypridina luciferin analog (FCLA)-1O2 can be modified with the presence of HSA. The conventional CL technique uses a quenching effect of HSA for its quantitative measurement. We are reporting here that the CL intensity can be enhanced, rather than quenched, by the addition of HSA. The CL signal can be linearly correlated with the HSA concentration over a clinically interesting range of 5 x 10(-9) - 8 x 10(-8) mol L(-1), with a detection limit of 2.5 x 10(-9) mol L(-1). The determination result was consistent with that obtained from conventional methods. One possible mechanism of HSA detection technique using CL enhancement approach is discussed. Intermolecular energy transfer in chemiluminescence systems and changes of microenvironment are likely to be contributors of the CL enhancement with HSA.     

Micellar electrokinetic chromatography-chemiluminescent detection of biogenic amines using N-(4-aminobutyl)-N-ethylisoluminol as derivatization reagent and trivalent copper chelate as chemiluminescence enhancer

A facile, sensitive and universal method was established for analysis of biogenic amines using micellar electrokinetic chromatography coupled with chemiluminescent (CL) detection. It was found that diperiodatocuprate (III) (K₅[Cu(HIO₆)₂], DPC), a transition metal chelate at unstable high oxidation state, could effectively enhance the reaction between luminol-type compound and hydrogen peroxide, to produce very strong CL signal. In addition, triethylamine was found to be able to effectively improve the yield of the derivatization reaction between biogenic amines and a luminol-type derivatization reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). Based on these facts, three biogenic amines were pre-column derivatized with ABEI, and post-column detected using high sensitive luminol-hydrogen peroxide-DPC CL system. Since the background was quite low, and the signal was quite strong, a considerable improved sensitivity was obtained. The presented method had been successfully applied to simultaneously analyze glycine, proline and phenylalanine with the detection limits (S/N = 3) of 0.030 μmol L⁻¹, 0.23 μmol L⁻¹ and 0.21 μmol L⁻¹, respectively. To evaluate its potential application value, glycine in saliva and urine samples was detected using this method, and satisfied results were obtained. This approach can be further extended to detection of many other compounds such as peptides and drugs by using luminol-type derivatization reagent.     

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol?L^?1 for all B19 genotype oligonucleotides and 650 pmol?L^?1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

基于拟糖蛋白的电化学阻抗生物传感器用于糖与蛋白质相互作用的研究

以D-甘露糖和伴刀豆球蛋白(ConA)的相互作用为研究对象,利用美拉德反应将D-甘露糖共价结合到负载蛋白牛血清蛋白(BSA)的表面形成拟糖蛋白,然后将拟糖蛋白固定到玻碳电极表面,以拟糖蛋白表面的D-甘露糖为分子识别物质,构建了检测伴刀豆球蛋白(ConA)的电化学阻抗传感器。拟糖蛋白制备过程简单,D-甘露糖负载量大,在空间中提供多个结合位点,因此能与ConA形成多价复合物,提高了传感器的灵敏度。该传感器的响应值与ConA浓度的对数在5.0×10-11~5.0×10-9mol/L之间呈良好的线性关系,检出限为1.7×10-11mol/L,D-甘露糖和ConA之间的结合常数为2.6×106L/mol。该方法简单,可适用于不同糖和蛋白质相互作用的研究,为构建高灵敏度的电化学阻抗传感器提供了新思路。     

Time-resolved luminescent lateral flow assay technology

We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ng mL⁻¹ in serum with a linear response from 0.2 to 200 ng mL⁻¹ CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.     

Determination of arsenate by sorption pre-concentration on polystyrene beads packed in a microfluidic device with chemiluminescence detection

A highly sensitive chemiluminescence (CL) method for the determination of arsenate in water based on a simple microfluidic device was developed. The method was based on sorption pre-concentration of arsenate as a form of vanadomolybdoarsenate heteropoly acid (VMoAs-HPA) ion-paired with hexadecyltrimethylammonium bromide on the surface of polystyrene beads packed in a microfluidic device monitored by chemiluminescence detection. The composition of the VMoAs-HPA complex was studied by varying the concentrations of ammonium molybdate, ammonium vanadate and sulfuric acid with a variable-size simplex optimization process, of which the optimum concentrations were 6.3 x 10(-5), 5.0 x 10(-6) and 1.0 x 10(-2) M, respectively. In this work, 1.0 x 10(-3) M ethylenediaminetetraacetic acid was added to all work solutions to remove the interferences of the other metal ions on the CL detection. The integration of sorption pre-concentration not only increased the detection sensitivity but also eliminated the interference from phosphate and chromate. The calibration plot was linear from 1.0 x 10(-7) to 5.0 x 10(-5) M As(v). The limit of detection was 8.9 x 10(-8) M As(v) (S/N = 3). The time required for one analysis run was as short as 5 min. The relative standard deviation was 5.9% (n = 9). This method was successfully applied to the determination of arsenate in mineral-, drinking- and tap-water samples.