Integrated Fluorescence Detection Research Articles

PEI-Mediated Assembly of Fe3O4 onto SiO2-Encapsulated CsPbBr3 for Highly Sensitive Fluorescent Lateral Flow Immunoassay.

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method's sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL-1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL-1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL-1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation.

Sense and Shoot: Multifunctional CaO2-loaded Cu-MOF nanosheets for integrated fluorescence detection and fenton-like degradation of tetracycline

Simultaneous accomplishment of sensitive detection and efficient degradation of antibiotics residues exposed in the aqueous environments has proved to be a difficult endeavor. Here, multifunctional composite CaO2-loaded Cu-MOF nanosheets (CaO2@Cu-MOF NSs) was elaborately designed and synthesized. Triggered by H+, the composite is not only capable of producing Ca2+ as the signal factor for tetracycline (TC) detection by activating the fluorescence emission of calcein, but also capable of self-supplying H2O2 and Cu2+ to construct a novel Fenton-like oxidation and O2 activation system for TC degradation based on Cu2+/Cu+ cycle. Under the synergy of target cycling and nano-enhancement, the sensing system exhibited great TC detection performance with a linear range of 0.001 to 100 ng mL−1 and detection limit of 11.8 fg mL−1. At the same time, the degradation system exhibited favorable degradation performance under the systematic action of Fenton-like and O2-activated oxidation. Nearly 80 % of the TC residues could be degraded within 10 min, and more than 95 % within 60 min. The constructed system was successfully applied to detect and degrade exposed TC in actual water samples in an integrated manner, demonstrating great promise for practical applications.

Extensible LED-Induced Integrated Fluorescence Detection Module for Quantitative Analysis of Lucigenin Concentration

We developed an extensible LED-induced fluorescence detection module with a highly integrated and ultra-compact structure. A target-oriented design methodology was used to demonstrate the module’s optimal design. Lucigenin solution was used as a test sample in evaluation trials to demonstrate the module’s quantitative fluorescence detection capability. Results showed that the integrated module has an outstanding linear response in the range of 0–1 μmol·L−1, with sensitivity and limit of detection (LOD) of 0.1692 V/μmol·L−1 and 0.03 μmol·L−1, respectively. Statistical analyses showed that our integrated module has extremely high repeatability and accuracy, i.e., the values of Pearson’s correlation coefficient and root-mean-square error exceeded 0.9995 and 1.8‰, respectively. More importantly, the integrated module possesses favorable extensibility and can realize on-demand rapid fluorescence-signal detection of other targets using appropriate parameter combinations. This module offers new opportunities for reliable, cost-effective and easy-to-use fluorescence-signal detection, especially in resource-constrained fluorescence detection applications.

Real-time isothermal nucleic acid amplification detection in resource-limited settings: a description of an open-source miniature fluorimeter

BackgroundThe detection and quantification of nucleic acids traditionally relies on PCR, which requires the use of expensive thermocyclers with integrated fluorescence detection of amplicons. While isothermal nucleic acid amplification technologies eliminate the need for thermocycling, fluorescence-based detection of products is still required for real-time, quantitative readout. Several commercially available isothermal heaters with integrated fluorescence detection exist, but the cost of these devices remains a significant barrier to the use of isothermal nucleic acid amplification in low-resource settings. A low-cost device for nucleic acid amplification detection could be used to contain the spread of the SARS-CoV-2 virus during the current pandemic as well as other disease targets in the future. Increasing COVID-19 testing capacity is particularly relevant when variations of the virus that appear to be more infectious are spreading rapidly and efforts to scale up delivery of COVID-19 vaccines are in the early stages. Here, we describe a modular, low-cost fluorimeter for use in resource-limited settings that lack access to and infrastructure for traditional testing platforms. MethodsWe present a fluorimeter that is constructed from off-the-shelf components enclosed in a compact 3D printed housing that removes the need for specialised optical alignment. The fluorimeter is designed to be placed atop a commercially available heat block holding a PCR-tube and was optimised to detect fluorescein (FITC) dye. The modular design allows it to be easily adapted for use with other dyes commonly used as reporters in nucleic acid amplification reactions. FindingsClinical applicability of the system was demonstrated using the system to measure amplification with two different isothermal amplification technologies with significantly different temperature, volume, and fluorescence requirements: recombinase polymerase amplification (RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). RPA detection was demonstrated with a positive control DNA provided in a commercial kit while RT-LAMP detection of clinically meaningful levels of SARS-CoV-2 RNA was demonstrated in a custom RT-LAMP assay. In a preliminary analysis of nasopharyngeal swab samples from 74 individuals, the custom RT-LAMP assay showed a positive agreement of 92·3% and a negative agreement of 91·4% with the SARS-CoV-2 RT-qPCR test developed by the US Centers for Disease Control and Prevention. InterpretationThis open-source system was designed to improve accessibility to nucleic acid testing with isothermal amplification methods. The system can be easily produced using only a low-end 3D printer. Moreover, the use of single-board computers obviates the need to produce printed circuit boards. At a time when global supply chains are at their most fragile, open-source diagnostic equipment has the potential to reduce pandemic-related health inequities. FundingInternational AIDS vaccine Initiative IAVI and USAID: award AID-OAA-A16-00032.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.

Microfluidics-based immunoassays by using an integrated fluorescence detection system

This manuscript presents an integrated fluorescence detection system including a disposable microfluidic chip integrated with an optical fiber for fluorescence immunoassays. When fluid samples and reagents were transported to the microfluidic chip by a MEMS-based micropump, the fluorescent light, which was induced by the excitation light from an LED coupled into the detection area of the microchannel through an optical fiber, was detected by a PMT. The system was able to detect from 1 to 1,000 μg/L fluorescein with a correlation coefficient of 0.966. In order to demonstrate the capability of the developed system further, human IgG was measured by the dual-antibody sandwich method by using this detection system. Several critical parameters such as different surface modification methods for antibody immobilization and various primary antibody concentrations of the sandwich immunoassays were optimized. Under the optimal condition, the detection range of human IgG was 0.16–20 mg/L with a correlation coefficient of 0.9802. The integrated fluorescence detection system has the potential to be used in the field of on-line point-of-care clinical testing.

Autonomous magnetically actuated continuous flow microimmunofluorocytometry assay

This article presents a microfluidic device which integrates autonomous serial immunofluorocytometry binding reactions of cytometric beads with fluorescence detection and quantification in a continuous flow environment. The microdevice assay is intended to alleviate the extensive benchwork and large sample volumes used when conducting traditional immunoassays, without requiring complex external controls. The technology is based on the miniaturization and automation of the serial processing steps of an antigen sandwich immunoassay, with integrated fluorescence detection using paramagnetic microbeads. The continuous flow design may enable temporal tracking of time-varying protein concentrations in a continuously infused sample for clinical applications, specifically for monitoring inflammation marker proteins in blood produced during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures. The device operation was first validated via a single incubation device which measured the concentration of a fluorescently labeled biotin molecule using streptavidin-coated paramagnetic cytometric beads. Subsequently, a dual incubation device was tested with samples of the anaphylatoxin complement protein C3a, and was shown to be capable of differentiating between samples at typical systemic concentrations of the protein (1-5 mug/ml), with very low sample usage (<6 mul/h). It is believed that this continuous flow, automated microimmunosensor technology will be a platform for high sample rate immunoassays capable of tracking and more thoroughly characterizing the systemic inflammation process, and may aid in the development of better treatment options for systemic inflammation during and after CPB.

Integration of optical fiber light guide, fluorescence detection system, and multichannel disposable microfluidic chip

A combination of fluorescence detection and microfluidic technology provides promising applications in life sciences. A prototype of an integrated fluorescence detection system and optical fiber light guide on a laminate-based multichannel microfluidic chip has been developed and tested. A blue LED, plastic optical fiber, photodiode, Mylar and PMMA, and fluorescein and BSA-FITC were used as an excitation source, light coupler and guide, detector, microfluidic substrate and sample, respectively. The results show that the system is capable of detecting weak fluorescence emission from a fluorescein solution at concentration down to 0.01 ng/ml, and gives linear response. The results were also reproducible, and no cross-talk between adjacent channels was observed. The test using BSA as a model analyte demonstrates its feasibility for on-chip immunosensor applications. The performance and applications can be developed further. This prototype can be used as a platform to develop a simple and compact bio-fluorescence detection system integrated with an inexpensive and disposable multichannel microfluidic chip for biomedical devices.

High-sensitivity integrated fluorescence analysis for microfluidic lab-on-a-chip

Microfluidics studies the behavior of fluids at the microscale and the design of systems to take advantage of such behavior. A multidisciplinary field encompassing physics, chemistry, engineering, and biotechnology, microfluidics integrates sensors, actuators, and other electronics to create new applications.1–3 Importantly, new principles of fluid manipulation have enabled detection and handling of nanoliter fluid samples. In recent years, these principles have been applied to the development of lab-on-a-chip (LOC) systems.4, 5 A substantial challenge for such miniaturized systems—the size of a credit card—for point-of-care and on-site analysis lies in developing their ability to effectively detect analytes of interest. Small sample volumes and low concentrations common to microfluidic LOCs make high-sensitivity detection of critical importance. While conventional microscopy tools can easily meet these demands in a laboratory, developing portable systems requires integrated miniaturized detectors. Fluorescence is one of the most commonly used analytic techniques in the biosciences. It is based on emission of a dye at one wavelength when it is illuminated by another, shorter wavelength. Lack of integrated fluorescence detection is a major roadblock for many biotechnological assays in portable LOC format. In a typical microfluidic fluorescence immunoassay assay, fluorescently labeled antibodies are used to tag a specific antigen (e.g., bacteria, viruses, or other organic molecules of interest) in a microfluidic device. The excitation light stimulates the dye to fluoresce, and the characteristic emission wavelength, or color, is observed through a filter that suppresses excitation light, enbling the dye emission to be seen clearly. As shown in Figure 1, the orange light represents the dye emission and the green light Figure 1. Schematic of a lab-on-a-chip (LOC) for fluorescence analysis. PDMS: Poly(dimethylsiloxane).

An integrated fluorescence detection system for lab-on-a-chip applications

We present a low-cost miniaturized fluorescence detection system for lab-on-a-chip applications with a sensitivity in the low nanomolar range; a built-in lock-in amplifier enables measurements under ambient light.

A novel concept of the integrated fluorescence detection system and its application in a lab-on-a-chip microdevice

In the paper, an innovative concept of fabrication of lab-on-a-chip (LOC) analytical microsystems, is presented. One of the major trends in design and fabrication of the LOC devices is integration of microfluidic separation part of the device with its detection optics. In our paper, we report a novel approach to this problem. As a substrate material for our LOC device, we choose soda lime glass plates in which we prepare channel optical waveguides, with the use of an ion exchange technique. Then, the microfluidic system is fabricated in the same substrate utilising standard photolithography and wet etching methods. As a result, we obtain a truly integrated, monolithic microsystem, containing both microchannels and microoptics, fabricated in the same substrate. The waveguiding optics is used to excite the fluorescence in the microchannels, while for the fluorescent beam collection, various approaches are tested: waveguide, free space and fibre optics. The quantitative results of the fluorescence experiments are also presented, namely calibration curves and level of detection values. Finally, an exemplary application of the system is demonstrated for the separation and detection of the mixture of Cy3 fluorescent dye and Cy3 tagged streptavidine.

Fabrication of an integrated PDMS microchip incorporating an LED-induced fluorescence device

A microfluidic device with an integrated fluorescence detection system has been developed in order to miniaturize the entire analytical system. A blue or green light-emitting diode (LED) and an optical fiber were mounted in a polydimethylsiloxane-based microchip. The performance of this device was evaluated by microchip electrophoresis. When a green LED was used as the light source, the calibration curve of Sulforhodamine-101 was linear over the range 1-100 microM. The detection limit was found to be 600 nM (240 amol) for a S/N ratio of 3. When using a blue LED, the calibration curve of Fluorescein was linear over the range 0.2-100 microM. The detection limit was estimated to be 120 nM (50 amol) (S/N=3). The detection sensitivity per unit power was comparable to that of LIF. The RSD values for the migration time, peak height and peak area were 0.74, 7.18 and 9.45%, respectively. The integrated microfluidic device was successfully used to determine amino acid derivatives.

Towards microalbuminuria determination on a disposable diagnostic microchip with integrated fluorescence detection based on thin-film organic light emitting diodes

As a first step towards a fully disposable stand-alone diagnostic microchip for determination of urinary human serum albumin (HSA), we report the use of a thin-film organic light emitting diode (OLED) as an excitation source for microscale fluorescence detection. The OLED has a peak emission wavelength of 540 nm, is simple to fabricate on flexible or rigid substrates, and operates at drive voltages below 10 V. In a fluorescence assay, HSA is reacted with Albumin Blue 580, generating a strong emission at 620 nm when excited with the OLED. Filter-less discrimination between excitation light and generated fluorescence is achieved through an orthogonal detection geometry. When the assay is performed in 800 microm deep and 800 microm wide microchannels on a poly(dimethylsiloxane)(PDMS) microchip at flow rates of 20 microL min(-1), HSA concentrations down to 10 mg L(-1) can be detected with a linear range from 10 to 100 mg L(-1). This sensitivity is sufficient for the determination of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease (clinical cut-off levels: 15-40 mg L(-1)).

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications.

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.

Evaluation of (In,Ga)N Films as Optical Absorption Filters for Application in Integrated Fluorescence Detection Micro-Bioanalytical Systems

AbstractFluorescence detection in integrated micro-bioanalytical systems requires the selective exclusion of light from the excitation source (typically blue or UV) and transmission of the longer wavelength fluorescence signal. In the present research, the application of (In,Ga)N alloy thin films as optical filters for absorption of blue (470 nm) and transmission of green (530 nm) has been evaluated. Absorption spectra, photoluminescence (PL) and Rutherford backscattering spectroscopy (RBS) results are presented for 200-400 nm (In,Ga)N thin films grown by MOCVD on sapphire substrates with 2-4 μm GaN buffer layers. A sigmoidal function was used to model the absorption coefficient of (In,Ga)N as a function of energy, Eg, and the broadening (ΔE) associated with the Urbach tail. Experimental data showed that, as expected, the absorption band edge for (In,Ga)N films broadened with increasing InN mole%. An increase in ΔE of 35 meV was observed when the InN mole% was increased from 10 to 16%. The sigmoidal function model provided a good fit to the experimental data, which allowed the experimental data to be extrapolated to higher InN concentrations. Based on this analysis, it is predicted that a 5 μm thick In0.22Ga0.78N film should transmit 50% of green light (530 nm) and only 3.29 x 1 0-4 of blue light (470 nm). The (In,Ga)N films that have been evaluated range in InN mole% from ~2-16%. The higher InN mole% samples (10-16%) can successfully filter lower wavelengths (e.g., 400 nm). For a filter application, it is also important that photoluminescence be effectively suppressed. In MQW structures, high PL intensity is often obtained by means of InN segregation (InN-rich nanoclusters). But for our evaluation with 200-400 nm (In,Ga)N films, relaxation of coherency strain results in large densities of dislocations, and correspondingly low PL intensity.