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Mixing during Trapping Enabled a Continuous-Flow Microfluidic Smartphone Immunoassay Using Acoustic Streaming
... How-ever, the amount of fluid used in such procedures is not acceptable for clinical applications. Acoustic waves generated by linear IDTs driven with a resonance frequency of 390 MHZ trap the fluorescent polystyrene particles diluted with PBS and mixed with immunocomplexes by the acoustic stream [73]. In a prostate-specific antigen (PSA) detection system, a wide dynamic response range from 0.3 ng/mL to 10 ng/mL was obtained with a detection limit of 0.2 ng/mL in a 10 µL sample [73]. ...
... Acoustic waves generated by linear IDTs driven with a resonance frequency of 390 MHZ trap the fluorescent polystyrene particles diluted with PBS and mixed with immunocomplexes by the acoustic stream [73]. In a prostate-specific antigen (PSA) detection system, a wide dynamic response range from 0.3 ng/mL to 10 ng/mL was obtained with a detection limit of 0.2 ng/mL in a 10 µL sample [73]. In addition to the mixture of liquids in a continuous flow, mixtures in the form of droplets are also frequently used methods in biological applications. ...
... A simple view of an active micromixer device[73], Copyright 2021, ACS Publications.(a) Acoustic Field-Driven Micromixers ...
Both passive and active microfluidic chips are used in many biomedical and chemical applications to support fluid mixing, particle manipulations, and signal detection. Passive microfluidic devices are geometry-dependent, and their uses are rather limited. Active microfluidic devices include sensors or detectors that transduce chemical, biological, and physical changes into electrical or optical signals. Also, they are transduction devices that detect biological and chemical changes in biomedical applications, and they are highly versatile microfluidic tools for disease diagnosis and organ modeling. This review provides a comprehensive overview of the significant advances that have been made in the development of microfluidics devices. We will discuss the function of microfluidic devices as micromixers or as sorters of cells and substances (e.g., microfiltration, flow or displacement, and trapping). Microfluidic devices are fabricated using a range of techniques, including molding, etching, three-dimensional printing, and nanofabrication. Their broad utility lies in the detection of diagnostic biomarkers and organ-on-chip approaches that permit disease modeling in cancer, as well as uses in neurological, cardiovascular, hepatic, and pulmonary diseases. Biosensor applications allow for point-of-care testing, using assays based on enzymes, nanozymes, antibodies, or nucleic acids (DNA or RNA). An anticipated development in the field includes the optimization of techniques for the fabrication of microfluidic devices using biocompatible materials. These developments will increase biomedical versatility, reduce diagnostic costs, and accelerate diagnosis time of microfluidics technology.
... Audio amplifier circuit generated sine waves with frequencies up to 70 kHz, which caused a flow rate up to 12 µL/min. Chen X. et al. presented a novel approach for particle trapping, based on the acoustofluidic approach with smartphone-based detection mechanism [131]. The proposed system was tested for PSA detection in both the buffer and serum and achieved a low detection limit: 0.2 ng/mL, and a large dynamic response range of 0.3 to 10 ng/mL. ...
... With the gradual modernization in the field of electronics, the size of such devices is rapidly decreasing, which in the near future, may lead to a complete replacement of passive flow control mechanisms in microfluidics. [128,[130][131][132] Current developments in the field of microfluidics have led to the creation of compact all-in-one systems. We suppose that novel microfluidic systems regardless of the fluid flow control will be created to be as compact as possible. ...
This review is an account of methods that use various strategies to control microfluidic flow control with high accuracy. The reviewed systems are divided into two large groups based on the way they create flow: passive systems (non-mechanical systems) and active (mechanical) systems. Each group is presented by a number of device fabrications. We try to explain the main principles of operation, and we list advantages and disadvantages of the presented systems. Mechanical systems are considered in more detail, as they are currently an area of increased interest due to their unique precision flow control and “multitasking”. These systems are often applied as mini-laboratories, working autonomously without any additional operations, provided by humans, which is very important under complicated conditions. We also reviewed the integration of autonomous microfluidic systems with a smartphone or single-board computer when all data are retrieved and processed without using a personal computer. In addition, we discuss future trends and possible solutions for further development of this area of technology.
... For example, magnetic-based microfluidic mixing was investigated where a fluid of dispersed magnetic particles or rods inside the chamber is actuated by an external magnet, 48,49 but this requires a closely attached magnetic actuator and the magnetic beads can interfere with the immunoassay performance while limiting the optical readout through the chamber. Moreover, acoustic actuation of embedded microstructures, [50][51][52] vibrating structures 53,54 or trapped microbubbles 55,56 has been investigated for microfluidic mixing which can require a lengthy microfabrication process, and the efficient trapping of the microbubbles inside the chamber throughout the full experiment, and possibly the need for high-frequency matching and signal generators. 57 Furthermore, electro-kinetic techniques such as the ACelectrothermal technique have been used for enhancing the microfluidic mixing and immunoassays, but it requires the microfabrication of the actuating electrodes and, if not optimized, can generate relatively high temperatures and lead to electrolysis. ...
The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming. Here, we report both by numerical simulation and experiments on the beneficiary effect of acoustic agitation on the uniformity of immunostaining in large-size and thin microfluidic chambers. Moreover, we investigate by numerical simulation the impact of reducing the incubation times and the concentrations of the biochemical detection reagents on the obtained immunoassay signal. Finally, acoustofluidic mixing was successfully used to reduce by 80% the incubation time of the Her2 (human epidermal growth factor receptor 2) and CK (cytokeratins) biomarkers for the spatial immunostaining of breast cancer cell pellets, or reducing their concentration by 66% and achieving a higher signal-to-background ratio than comparable spatially resolved immunostaining with static incubation.
... 1−3 Applications of surface acoustic waves (SAWs) in microfluidic platforms (often called as acoustofluidics) have recently gained great interest for manipulating fluids, microparticles/cells, in either a digital form (sessile droplet) or a continuous flow (inside a channel/chamber). 4−8 These acoustofluidic devices have shown remarkable potential in mixing, 9,10 pumping, 11 jetting, 12,13 and atomizing 14,15 of fluids on the microscale and applications in the fields of biomedicine and chemistry for non-invasive and contactless manipulation, with low cost, good biocompatibility, and conserved cell viability and proliferation capacity. 16−20 SAW acoustofluidic devices are generally fabricated by pattering IDTs on a piezoelectric substrate such as a LiNbO 3 or a piezoelectric film on a silicon or Al plate substrate, 21−23 which converts the radio frequency (RF) signal into SAWs propagating along the surface of the substrate. ...
Surface acoustic wave (SAW)-based acoustofluidic devices have shown broad applications in microfluidic actuation and particle/cell manipulation. Conventional SAW acoustofluidic device fabrication generally includes photolithography and lift-off processes and thus requires accessing cleanroom facilities and expensive lithography equipment. In this paper, we report a femtosecond laser direct writing mask method for acoustofluidic device preparation. By micromachining of steel foil to form the mask and direct evaporation of metal on the piezoelectric substrate using the mask, the interdigital transducer (IDT) electrodes of the SAW device are generated. The minimum spatial periodicity of the IDT finger is about 200 μm, and the preparation for LiNbO3 and ZnO thin films and flexible PVDF SAW devices is verified. Meanwhile, we have demonstrated various microfluidic functions, including streaming, concentration, pumping, jumping, jetting, nebulization, and particle alignment using the fabricated acoustofluidic (ZnO/Al plate, LiNbO3) devices. Compared to the traditional manufacturing process, the proposed method omits spin coating, drying, lithography, developing, and lift-off processes and thus has advantages of simple, convenient, low cost, and environment friendliness.
... Sequential micromixing between multiple parallel streams is still challenging. By contrast, active micromixing is driven by external energy sources, such as magnetic fields [20,21], acoustic energy [22,23], electric stimulation [24][25][26][27], and optical fields [28]. An active mixer enables fast and homogeneous micromixing within a simple channel and in a short time. ...
Multi-fluid micromixing, which has rarely been explored, typically represents a highly sought-after technique in on-chip biochemical and biomedical assays. Herein, we propose a novel micromixing approach utilizing induced-charge electroosmosis (ICEO) to implement multicomplex mixing between parallel streams. The variations of ICEO microvortices above a sinusoidally shaped floating electrode (SSFE) are first investigated to better understand the microvortex development and the resultant mixing process within a confined channel. On this basis, a mathematical model of the vortex index is newly developed to predict the mixing degree along the microchannel. The negative exponential distribution obtained between the vortex index and mixing index demonstrates an efficient model to describe the mixing performance without solving the coupled diffusion and momentum equations. Specifically, sufficient mixing with a mixing index higher than 0.9 can be achieved when the vortex index exceeds 51, and the mixing efficiency reaches a plateau at an AC frequency close to 100 Hz. Further, a rectangle floating electrode (RFE) is deposited before SSFE to enhance the controlled sequence for three-fluid mixing. One side fluid can fully mix with the middle fluid with a mixing index of 0.623 above RFE in the first mixing stage and achieve entire-channel mixing with a mixing index of 0.983 above SSFE in the second mixing stage, thereby enabling on-demand sequential mixing. As a proof of concept, this work can provide a robust alternative technique for multi-objective issues and structural design related to mixers.
... Here, we qualitatively discuss the influence of the boundary conditions and the force status of the trapped particles by simulation. Once the device was fixed, the strength and distribution of the acoustic streaming were mainly determined by the geometry of the microchannel 59,63,64 . We then calculated the distribution of the acoustic field in 2D models under different geometric confinements, as shown in Supplementary Fig. S3. ...
At the single-cell level, cellular parameters, gene expression and cellular function are assayed on an individual but not population-average basis. Essential to observing and analyzing the heterogeneity and behavior of these cells/clusters is the ability to prepare and manipulate individuals. Here, we demonstrate a versatile microsystem, a stereo acoustic streaming tunnel, which is triggered by ultrahigh-frequency bulk acoustic waves and highly confined by a microchannel. We thoroughly analyze the generation and features of stereo acoustic streaming to develop a virtual tunnel for observation, pretreatment and analysis of cells for different single-cell applications. 3D reconstruction, dissociation of clusters, selective trapping/release, in situ analysis and pairing of single cells with barcode gel beads were demonstrated. To further verify the reliability and robustness of this technology in complex biosamples, the separation of circulating tumor cells from undiluted blood based on properties of both physics and immunity was achieved. With the rich selection of handling modes, the platform has the potential to be a full-process microsystem, from pretreatment to analysis, and used in numerous fields, such as in vitro diagnosis, high-throughput single-cell sequencing and drug development.
... Trapping, separation, and transportation of particles and mixing of fluids are the key functionalities of acoustic micro actuators. As a result, acousticactuated microfluidic platforms have been applied to live science and biomolecular analysis, such as cancer diagnosis [240], protein, and miRNA detection [241]. Recently, Chen et al. reported a microfluidic immunoassay based on acoustic streaming tweezers for the detection of prostate-specific antigen [242]. ...
Surface plasmonic resonance (SPR) has been a corner stone for approaching single molecular detection due to its high-sensitivity capability and simple detection mechanism, and has brought major advancements in biomedicine and life science technology. Over decades, the successful integration of SPR with versatile techniques has been demonstrated. However, several crucial limitations have hindered this technique for practical applications, such as long detection time and low overall sensitivity. This review aims to provide a comprehensive summary of existing approaches in enhancing the performance of SPR sensors based on “passive” and “active” methods. Firstly, passive enhancement is discussed from a material aspect, including signal amplification tags and modifications of conventional substrates. Then, the focus is on the most popular active enhancement methods including electrokinetic, optical, magnetic, and acoustic manipulations that are summarized with highlights on their advantageous features and ability to concentrate target molecules at the detection sites. Lastly, prospects and future development directions for developing SPR sensing towards a more practical, single molecular detection technique in the next generation are discussed. This review hopes to inspire researchers’ interests in developing SPR-related technology with more innovative and influential ideas.
... The immunoassay chip integrates various immunodetection steps in a tiny device. Among the integration techniques, the microfluidics-based immunoassay chip is a popular device because of the small liquid volumes, rapid turnaround time, and high portability offered by microfluidics [1][2][3][4][5][6][7]. Using beads as carriers, the surface area available for antigenantibody binding is greatly increased, thereby enhancing the signal and shortening the reaction time [8][9][10][11][12][13][14][15]. ...
In a biomedical diagnosis with a limited sample volume and low concentration, droplet-based microfluidics, also called digital microfluidics, becomes a very attractive approach. Previously, our group developed a magnetic-beads-based digital microfluidic immunoassay with a bead number of around 100, requiring less than 1 μL of sample volume to achieve a pg/mL level limit of detection (LOD). However, the bead number in each measurement was not the same, causing an unstable coefficient of variation (CV) in the calibration curve. Here, we investigated whether a fixed number of beads in this bead-based digital microfluidic immunoassay could provide more stable results. First, the bead screening chips were developed to extract exactly 100, 49, and 25 magnetic beads with diameters of less than 6 μm. Then, four calibration curves were established. One calibration curve was constructed by using varying bead numbers (50–160) in the process. The other three calibration curves used a fixed number of beads, (100, 49, and 25). The results indicated that the CVs for a fixed number of beads were evidently smaller than the CVs for varying bead numbers, especially in the range of 1 pg/mL to 100 pg/mL, where the CVs for 100 beads were less than 10%. Furthermore, the calculated LOD, based on the composite calibration curves, could be reduced by three orders, from 3.0 pg/mL (for the unfixed bead number) to 0.0287 pg/mL (for 100 beads). However, when the bead numbers were too high (more than 500) or too low (25 or fewer), the bead manipulation for aggregation became more difficult in the magnetic-beads-based digital microfluidic immunoassay chip.
... This is because the antibody-antigen binding is more limited by transport of the analyte to the antibody rather than the binding kinetics. Therefore, mixing enhancement is usually required to improve the binding efficiency and to reduce the immunoassay time [21][22][23][24]. For POCT applications, the microfluidic chips or micro-total analytical systems (µ-TAS) are usually preferred due to their technical advantages such as low consumption of samples reagents, high sensitivity, reduced testing time etc. [25,26] However, in miniaturized fluidic devices, the fluid viscosity becomes dominant and the fluid mixing becomes difficult [27]. ...
Procalcitonin (PCT) is an important biomarker of bacterial infections. Detection of serum PCT is of great significance in diagnosis and therapy of a variety of diseases. In this work it is measured through immunoassay using antibody-conjugated fluorescent microspheres. To improve the binding efficiency of PCT antigen and antibody, an aeroelastic agitator is designed. Through alternate perturbations, it produces chaotic advection to enhance the fluid mixing. The influences of key design and operating factors are analyzed, including the geometrical parameters, the driving pressure, and the alternating period. Then the agitator is applied for PCT detection and its performance is compared with an ultrasonic mixer. Results show that the chaotic mixing significantly improves the capturing efficiency of PCT antigen, leading to a better detection consistency and reliability. The fluorescence intensity correlates fairly well with the serum PCT concentration in the range of 0.02 ng/ml - 49.56 ng/ml.
Sample preparation turns out to be one of the important procedures in complex sample analysis by affecting the accuracy, selectivity, and sensitivity of analytical results. However, the majority of the conventional sample preparation techniques still suffer from time-consuming and labor-intensive operations. These shortcomings can be addressed by reforming the sample preparation process in a microfluidic manner. Inheriting the advantages of rapid, high efficiency, low consumption, and easy integration, microfluidic sample preparation techniques receive increasing attention, including microfluidic phases separation, microfluidic field-assisted extraction, microfluidic membrane separation, and microfluidic chemical conversion. This review overviews the progress of microfluidic sample preparation techniques in the last 3 years based on more than 100 references, we highlight the implementation of typical sample preparation methods in the formats of microfluidics. Furthermore, the challenges and outlooks of the application of microfluidic sample preparation techniques are discussed.
Microfluidic technology has emerged as a promising tool in various applications, including biosensing, disease diagnosis, and environmental monitoring. One of the notable features of microfluidic devices is their ability to selectively capture and release specific cells, biomolecules, bacteria, and particles. Compared to traditional bulk analysis instruments, microfluidic capture-and-release platforms offer several advantages, such as contactless operation, label-free detection, high accuracy, good sensitivity, and minimal reagent requirements. However, despite significant efforts dedicated to developing innovative capture mechanisms in the past, the release and recovery efficiency of trapped particles have often been overlooked. Many previous studies have focused primarily on particle capture techniques and their efficiency, disregarding the crucial role of successful particle release for subsequent analysis. In reality, the ability to effectively release trapped particles is particularly essential to ensure ongoing, high-throughput analysis. To address this gap, this review aims to highlight the importance of both capture and release mechanisms in microfluidic systems and assess their effectiveness. The methods are classified into two categories: those based on physical principles and those using biochemical approaches. Furthermore, the review offers a comprehensive summary of recent applications of microfluidic platforms specifically designed for particle capture and release. It outlines the designs and performance of these devices, highlighting their advantages and limitations in various target applications and purposes. Finally, the review concludes with discussions on the current challenges faced in the field and presents potential future directions.
Accurate quantification of proprotein convertase subtilisin/kexin type 9 (PCSK9) in serum before and after the medication is helpful in grasping the evolution of PCSK9-related disease and evaluating the efficacy of PCSK9 inhibitors. Conventional approaches for PCSK9 quantification suffered from complicated operations and low sensitivity. By integrating stimuli-responsive mesoporous silica nanoparticles, dual-recognition proximity hybridization, and T7 exonuclease-assisted recycling amplification, a novel homogeneous chemiluminescence (CL) imaging approach was proposed for ultrasensitive and convenient immunoassay of PCSK9. Owing to the intelligent design and signal amplification property, the whole assay was conducted without separation and rinsing, significantly simplifying the procedure and eliminating the errors associated with the professional operation; meanwhile, it showed linear ranges over 5 orders of magnitude and detection limit as low as 0.7 pg mL-1. Parallel testing was allowed due to the imaging readout, which brought a maximum throughput of 26 tests h-1. The proposed CL approach was applied to analyze PCSK9 from hyperlipidemia mice before and after the intervention of the PCSK9 inhibitor. Serum PCSK9 levels in the model group and the intervention group could be distinguished efficiently. The results were reliable compared to commercial immunoassay results and histopathologic findings. Thus, it could facilitate the monitoring of the serum PCSK9 level and the lipid-lowering effect of the PCSK9 inhibitor, showing promising potential in bioanalysis and pharmaceuticals.
Over the past few decades, acoustofluidics, one of the branches of microfluidics, has rapidly developed as a multidisciplinary cutting edge research topic, covering many biomedical and bioanalytical applications. Acoustofluidics usually utilizes acoustic pressure and acoustic streaming effects to manipulate liquids and bioparticles. Acoustic manipulation using acoustic radiation force has been widely studied; however, with the recent development of new piezoelectric devices that enable faster acoustic streaming, particle manipulations using drag force induced by acoustic streaming have attracted more attention. Despite many review articles on acoustic radiation force-based acoustophoresis, acoustic streaming is less frequently covered. Here, we review the recent development of microscale acoustic streaming, especially high-frequency transducer-induced high-speed streaming, confinement and programed streaming, and acoustic streaming tweezers, which combine the acoustic radiation force and drag force to tackle the size limitations of conventional acoustic manipulations. A brief review of acoustic streaming theory and its generation is summarized. Recent progress in applying acoustic streaming for fluidic handling and bioparticle manipulations is reviewed. Representative applications of micro acoustic streaming are provided, and the key issues in these applications are analyzed. Finally, the future prospects of micro acoustic streaming in bioanalytical and biomedical applications are discussed.
Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.
Microfluidic chip technology is a technology platform that integrates basic operation units such as processing, separation, reaction and detection into microchannel chip to realize low consumption, fast and efficient analysis of samples. It has the characteristics of small volume need of samples and reagents, fast analysis, low cost, automation, portability, high throughout, and good compatibility with other techniques. In this review, the concept, preparation materials and fabrication technology of microfluidic chip are described. The applications of microfluidic chip in immunoassay, including fluorescent, chemiluminescent, surface-enhanced Raman spectroscopy (SERS), and electrochemical immunoassay are reviewed. Look into the future, the development of microfluidic chips lies in point-of-care testing and high throughput equipment, and there are still some challenges in the design and the integration of microfluidic chips, as well as the analysis of actual sample by microfluidic chips.
Point of care (POC) testing is of great importance in rapid diagnosis of disease, monitoring environmental pollutants, food safety analysis and other fields, especially in resource-limited settings. Microfluidic sensors combined with miniaturized portable detection devices are ideal for POC testing, which significantly reduce the reagent/sample consumption and shorten the analysis time without relying on laboratories and operators. Smartphone-based microfluidic sensors are suitable for POC detection because of their ability to high computational capability and data transferability, making them rapidly emerging as a potential substitute for traditional laboratory instruments. In this review, we introduced the functions of smartphones could achieve in the microfluidic sensing and summarized the detection routes involved in smartphone-based microfluidic sensors, including colorimetric, fluorescent, electrochemical and chemiluminescent sensing, together with thermal imaging and multiple-mode detection. An overview of recent applications in disease diagnosis, environmental monitoring and food safety detection fields was provided. Challenges and future perspectives were discussed.
Self-contained microfluidic platforms with on-chip integration of flow control units, microreactors, (bio)sensors, etc. are ideal systems for point-of-care (POC) testing. However, current approaches such as micropumps and microvalves, increase the cost and the control system, and it is rather difficult to integrate into a single chip. Herein, we demonstrated a versatile acoustofluidic platform actuated by a Lamb wave resonator (LWR) array, in which pumping, mixing, fluidic switching, and particle trapping are all achieved on a single chip. The high-speed microscale acoustic streaming triggered by the LWR in the confined microchannel can be utilized to realize a flow resistor and switch. Variable unidirectional pumping was realized by regulating the relative position of the LWR in various custom-designed microfluidic structures and adoption of different geometric parameters for the microchannel. In addition, to realize quantitative biomarker detection, the on-chip flow resistor, micropump, micromixer and particle trapper were also integrated with a CMOS photo sensor and electronic driver circuit, resulting in an automated handheld microfluidic system with no moving parts. Finally, the acoustofluidic platform was tested for prostate-specific antigen (PSA) sensing, which demonstrates the biocompatibility and applied potency of this proposed self-contained system in POC biomedical applications.
With the growing demand for early diagnosis of malignant tumor, ultrasensitive detection of cancer-related biomolecules with convenient operation is of great significance for timely treatment. In this study, we develop a robust and efficient analytical system, the aptamer-based chemiluminescent optical fiber immunosensor (aptamer-COFIS), for ultrasensitive detection of biomarkers. In the aptamer-COFIS system, target aptamer-primer probe is used as the bridge to combine antibody-antigen immunoreaction on the fiber surface with chemiluminescent signal amplification. The antibody-antigen (target)-aptamer sandwich-like reaction is coupled with rolling cycle amplification (RCA), which is capable to produce hundreds of binding sites to connect with the streptavidin-biotinylated horseradish peroxidase nanocomposites for signal amplification. Using prostate specific antigen (PSA), the most reliable and specific biomarker of prostate cancer, as the test sample, we show that the proposed strategy can greatly improve the sensor response with a wide linear range of 0.01-1000 pg/mL and an extremely low limit-of-detection of 3.2 fg/mL. Our results indicate that the proposed method presents high selectivity and good accuracy for ultrasensitive sensing biomarkers in complex clinical samples. Moreover, with additional advantages by using optical fiber as the ideal carrier of biorecognition elements and the transducer of chemiluminescent signal, the aptamer-COFIS system with a low-cost compact design is convenient for operation, making it suitable for cost-effective on-site and real time analysis of various biomarkers in early diagnosis of diseases.
A microfluidic platform for the integration of multi-step biological assays has been developed. The presented system is a unique instrument compatible with microfluidic chips for various applications based on bead manipulation. Two examples of microfluidic cartridges are presented here. The first one contains two rows of eight chambers (40 and 80 μL), six reagent inlets, eight testing solution (calibrators and samples) inlets and eight outlets to reproduce precisely each step of a biological assay. This configuration is versatile enough to integrate many different biological assays and save a lot of development time. The second architecture is dedicated to one specific protocol and is completely automated from the standard and sample dilutions to the optical detection. Linear dilutions have been integrated to prepare automatically a range of standard concentrations and outlets have been modified for integrated colorimetric detection. The technology uses pneumatically collapsible chambers to perform all the fluidic operations for a fully automated protocol such as volume calibrations, fluid transport, mixing, and washing steps. A programmable instrument with a software interface has been developed to adapt rapidly a protocol to this cartridge. As an example, these new microfluidic cartridges have been used to successfully perform an immunoassay for gluten detection in the dynamic range of 10-30 ppm with good sensitivity (2 ppm) and specificity.
Okadaic acid (OA) is one of the main virulence factors of diarrheal shellfish toxins (DSP), which can cause acute carcinogenic or teratogenic effects after ingestion of contaminated shellfish. Therefore, high-sensitivity and fast detection of OA is a key to preventing the occurrence of safety accidents. In this paper, we effectively established a smartphone-assisted microarray immunosensor combined with an indirect competitive ELISA (iELISA) for quantitative colorimetric detection of OA. To further improve the detection sensitivity and match the smartphone imaging, a novel graphene oxide (GO) composite probe was developed to realize the multi-stage signal amplification. The system exhibited a wide linear range for the detection of OA (0.02–33.6 ng·mL⁻¹) with low detection limit of 0.02 ng·mL⁻¹. The recovery of OA in spiked shellfish samples was in the range of 80%–103.5%, which indicates the good applicability of this biosensor. The whole detection system has advantages of simplicity, low cost, high sensitivity and portability, which is expected to be a powerful alternative tool for on-site detecting and early warning of the pollution of marine products.
Attachment of biorecognition molecules prior to microfluidic packaging is advantageous for many silicon biosensor-based lab-on-a-chip devices. This necessitates biocompatible bonding of the microfluidic cartridge, which, due to thermal or chemical incompatibility, excludes standard microfabrication bonding techniques. Here, we demonstrate a novel processing approach for a commercially available, two-step curable polymer to obtain biocompatible UVA-bonding of polymer microfluidics to silicon biosensors. Biocompatibility is assessed by UVA-bonding to antibody-functionalized ring resonator sensors and performing antigen capture assays while optically monitoring the sensor response. The assessments indicate normal biological function of the antibodies after UVA-bonding with selective binding to the target antigen. The bonding strength between polymer and silicon chips (non-biofunctionalized and biofunctionalized) is determined in terms of static liquid pressure. Polymer microfluidic cartridges are stored for more than 18 weeks between cartridge molding and cartridge-to-silicon bonding. All bonded devices withstand more than 2500 mbar pressure, far exceeding the typical requirements for lab-on-a-chip applications, while they may also be de-bonded after use. We suggest that these characteristics arise from bonding mainly through intermolecular forces, with a large extent of hydrogen bonds. Dimensional fidelity assessed by microscopy imaging shows less than 2% shrinkage through the molding process and the water contact angle is approximately 80°. As there is generally little absorption of UVA light (365 nm) in proteins and nucleic acids, this UVA-bonding procedure should be applicable for packaging a wide variety of biosensors into lab-on-a-chip systems.
Particle separation using surface acoustic waves (SAWs) has been a focus of ongoing research for several years, leading to promising technologies based on Lab-on-a-Chip devices. In many of them, scattering effects of acoustic waves on suspended particles are utilized to manipulate their motion by means of the acoustic radiation force (FARF). Due to viscous damping of radiated waves within a fluid, known as the acoustic streaming effect, a superimposed fluid flow is generated, which additionally affects the trajectories of the particles by drag forces. To evaluate the influence of this acoustically induced flow on the fractionation of suspended particles, the present study gives a deep insight into the pattern and scaling of the resulting vortex structures by quantitative three-dimensional, three component (3D3C) velocity measurements. Following the analysis of translationally invariant structures at the center of a pseudo-standing surface acoustic wave (sSAW) in Part I, the focus in Part II turns to the outer regions of acoustic actuation. The impact of key parameters on the formation of the outer vortices, such as the wavelength of the SAW λSAW, the channel height H and electrical power Pel, is investigated with respect to the design of corresponding separation systems. As a result of large gradients in the acoustic fields, broadly extended vortices are formed, which can cause a lateral displacement of particles and are thus essential for a holistic analysis of the flow phenomena. The interaction with an externally imposed main flow reveals local recirculation regions, while the extent of the vortices is quantified based on the displacement of the main flow.
Rapid detection and accurate analysis of trace samples is an important prerequisite for precision medicine. Here we integrated capillary with ultrasound to induce biomarkers enrichment in nanoliter samples, and developed a nanoliter sample enrichment analysis method for ultra-trace miRNA biosensing. The interaction between ultrasonic field and capillary provides a gradient ultrasound field, which is essential for the aggregation of functionalized microspheres along with the enrichment of specific biomarkers. The results indicated that the enrichment of the biomarkers effectively enhanced the fluorescence intensity, and the limit of detection reaches 7.8✕10⁻¹² M in 100 nL. Such integrated device can realize ultrasonic enrichment and visual analysis of target samples, and provides a new idea for rapid and highly sensitive detection of ultra-trace biomarkers in clinical diagnosis.
Acoustofluidics-assisted colorimetric method, a promising point-of-care test (POCT) strategy, has been applied in convenient detection of target molecules using the sensing nanomaterials, but remains a formidable challenge to design fluorescent probe with profuse color evolution, realizing sensitive, simple and accurate determination of analyte. Here, a novel triple-emission fluorescent probe is constructed through a simple blend of two ratiometric fluorescent probes (blue-organosilane-polymerized carbon [email protected]2 [email protected]/CdS quantum dots ([email protected]2@r) and blue-organosilane-polymerized carbon [email protected]2 [email protected]/CdS quantum dots ([email protected]2@g)) at the optimized volume ratio of 11:8. As signal reporters of the triple-emission probe, outer modified “r” and “g” will be quenched in sequence by target via two-step response strategy while the inner “b” as internal standard remains constant. When acoustic field is applied, the effect of acoustofluidics-based nanoparticles concentration makes triple-emission probe aggregate completely, accompanying fluorescence color signal being enhanced, sensitivity of colorimetric assay being improved and multicolor-variation (from orange to dark-goldenrod to dark-olive-green to sea-green to dark-cyan to final steel-blue) with the increase of analyte amount. For POCT demonstration, hemoglobin (Hb) is used as a model target and the captured fluorescent photos of triple-emission probe aggregates are analyzed by a chromaticity analysis application on the smartphone to calculate the red, green and blue values, which are used for accurate Hb quantification with the limit of detection of 1.99 mg/L. Whole test is finished within ∼12 min. The acoustofluidics-manipulated triple-emission probe biosensing strategy has also been applied to the rapid and visual quantitative detection of Hb in human blood.
At the single-cell level, cellular parameters, gene expression and function are assayed on an individual but not population-average basis. Essential to observing and analyzing the heterogeneity and behavior of these cells/clusters is the ability to prepare and manipulate individual. Here, we demonstrate a versatile microsystem, a stereo acoustic streaming tunnel, which is triggered by ultrahigh-frequency bulk acoustic waves and highly confined by a microchannel. We thoroughly analyze the generation and feature of stereo acoustic streaming to develop a virtual tunnel for observation, pretreatment and analysis of cells for different single-cell applications. 3D reconstruction, dissociation of clusters, selective trapping/release, in-situ analysis and pairing of single cells with barcode gel beads were demonstrated. In order to further verify the reliability and robustness of this technology in complex bio-samples, separation of circulating tumor cells based on both physics and immunity from undiluted blood was achieved. With the rich selection of handling modes, the platform has the potential to be a full-process microsystem from pretreatment to analysis and used in numerous fields, such as in vitro diagnosis, high-throughput single-cell sequencing and drug development.
At the single-cell level, cellular parameters, gene expression and function are assayed on an individual but not population-average basis. Essential to observing and analyzing the heterogeneity and behavior of these cells/clusters is the ability to prepare and manipulate individual. Here, we demonstrate a versatile microsystem, a stereo acoustic streaming tunnel, which is triggered by ultrahigh-frequency bulk acoustic waves and highly confined by a microchannel. We thoroughly analyze the generation and feature of stereo acoustic streaming to develop a virtual tunnel for observation, pretreatment and analysis of cells for different single-cell applications. 3D reconstruction, dissociation of clusters, selective trapping/release, in-situ analysis and pairing of single cells with barcode gel beads were demonstrated. In order to further verify the reliability and robustness of this technology in complex bio-samples, separation of circulating tumor cells based on both physics and immunity from undiluted blood was achieved. With the rich selection of handling modes, the platform has the potential to be a full-process microsystem from pretreatment to analysis and used in numerous fields, such as in vitro diagnosis, high-throughput single-cell sequencing and drug development.
Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/μL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.
Given the continuous and growing demand for point of care (POC) diagnostic tests, attention has been shifted toward integration and miniaturization of laboratory protocols into “sample‐in‐answer‐out” devices. Microfluidic technologies have been considered an ideal solution to address the requirements of POC diagnostics since many laboratory functions can be miniaturized and incorporated onto a single integrated chip. In this review, we summarize the advances of integrated microfluidic devices for POC diagnostics in the last 3 years. Particularly, we summarize current materials used for microfluidic chip fabrication, discuss the innovation of versatile integrated microfluidic devices, especially the strategies for simplifying sample preparation in manual or self‐driven systems, and new detection methods of microfluidic chips. In addition, we describe new integrated microfluidic devices for POC diagnostics of protein‐targeted immunodiagnostics, nucleic acid molecular tests, and small molecule metabolites analysis. We also provide future perspectives and current challenges for clinical translation and commercialization of these microfluidic technologies. Microfluidic platforms represent one of the most promising strategies for POC diagnostics because they eliminate the need for costly instruments with high training requirements by integrating several conventional laboratory protocols into single analytical diagnostic step. Here, we review several major advances from the past 3 years in integrated microfluidic devices for POC diagnostic testing.
Smartphone-based imaging devices (SIDs) have shown to be versatile and have a wide range of biomedical applications. With the increasing demand for high-quality medical services, technological interventions such as portable devices that can be used in remote and resource-less conditions and have an impact on quantity and quality of care. Additionally, smartphone-based devices have shown their application in the field of teleimaging, food technology, education, etc. Depending on the application and imaging capability required, the optical arrangement of the SID varies which enables them to be used in multiple setups like bright-field, fluorescence, dark-field, and multiple arrays with certain changes in their optics and illumination. This comprehensive review discusses the numerous applications and development of SIDs towards histopathological examination, detection of bacteria and viruses, food technology, and routine diagnosis. Smartphone-based devices are complemented with deep learning methods to further increase the efficiency of the devices.
Graphical Abstract
There has been a considerable development in microfluidic based immunodiagnostics over the past few years which has greatly favored the growth of novel point-of-care-testing (POCT). However, the realization of an inexpensive, low-power POCT needs cheap and disposable microfluidic devices that can perform autonomously with minimum user intervention. This work, for the first time, reports the development of a new microchannel capillary flow assay (MCFA) platform that can perform chemiluminescence based ELISA with lyophilized chemiluminescent reagents. This new MCFA platform exploits the ultra-high sensitivity of chemiluminescent detection while eliminating the shortcomings associated with liquid reagent handling, control of assay sequence and user intervention. The functionally designed microchannels along with adequate hydrophilicity produce a sequential flow of assay reagents and autonomously performs the ultra-high sensitive chemiluminescence based ELISA for the detection of malaria biomarker such as PfHRP2. The MCFA platform with no external flow control and simple chemiluminescence detection can easily communicate with smartphone via USB-OTG port using a custom-designed optical detector. The use of the smartphone for display, data transfer, storage and analysis, as well as the source of power allows the development of a smartphone based POCT analyzer for disease diagnostics. This paper reports a limit of detection (LOD) of 8 ng/mL by the smartphone analyzer which is sensitive enough to detect active malarial infection. The MCFA platform developed with the smartphone analyzer can be easily customized for different biomarkers, so a hand-held POCT for various infectious diseases can be envisaged with full networking capability at low cost.
Analysis on a single-cell basis is both fundamental and meaningful in biomedical research and clinical practice. Flow cytometry is one of the most popular approaches in this field with broad applications in cell sorting, counting, and identification of rare cells. However, the complicated design and bulky size of conventional flow cytometry have restricted their applications mainly in centralized laboratories. With the recent development of smartphone devices, smartphone-based cytometry has been explored and tested for single-cell analysis. Compared with traditional cytometers, smartphone-based cytometric biosensors are more suitable for point-of-care (POC) uses, such as on-site disease diagnosis and personal health monitoring. In this review article, the history of traditional flow cytometry is introduced, and advances of smartphone-enabled cytometry are summarized in detail based on different working principles. Representative POC applications of smartphone cytometers are also discussed. The achievements demonstrated so far illustrate the potential of smartphone-based cytometric devices to transform single-cell measurement in general, with a significant impact in POC diagnostics, preventive medicine, and cell biology. Keywords: Flow cytometry, Smartphone, Point-of-care diagnostics, Single-cell analysis, Imaging, Microfluidics
Bead-based immunosesnors have been intriguing the scientific community over the past decades due to their rapid and multiplexed capabilities in the detection of various biological targets. Nevertheless, their use in the detection of low-abundance analytes remains a continual challenge because of the limited number of active enrichment approaches. To this end, our research presents a delicate microbead enrichment by an optoelectrokinetic platform, followed by the detection of dual biomarkers for the sensitive screening of an eye disease termed diabetic retinopathy (DR). In this study, microbeads turned fluorescent as their surfaces formed sandwiched immunocomplexes in the presence of target antigens. Tiny fluorescent dots were then concentrated by the optoelectrokinetic platform for the enhancement of their signals. The signal rapidly escalated in 10 s, and the optimal limit of detection was nearly 100 pg/mL. For practical DR screening, two biomarkers, lipocalin 1 (LCN1) and vascular endothelial growth factor (VEGF), were used. Approximately 20 μL of analytes was collected from the tear samples of the tested patients. The concentrations of both biomarkers showed escalating trends with the severity of DR. Two concentration thresholds of LCN1 and VEGF that indicate proliferative DR were determined out of 24 clinical samples based on the receiver operating characteristic curves. For verification, a single-blinded test was conducted with additional clinical tear samples from five random subjects. The final outcome of this evaluation showed an accuracy of >80%. This noninvasive screening provides a potential means for the early diagnosis of DR and may increase the screening rate among the high-risk diabetic population in the future.
It is critical to reliably and rapidly detect multiple disease biomarkers in tiny liquid samples with high sensitivity to meet the growing demand for point-of-care diagnostics. This paper reports a microfluidic platform integrating magnetic-based single bead trapping in conjunction with acoustic micromixing for simultaneous detection of multiple cancer biomarkers within minutes. Individual beads retained by permalloy (NiFe81/19) microarray were used to capture biomarkers and facilitate the fluorescence identification. A numerical study indicates that the magnetic force keeping a bead in the trap is proportional to the thickness of the permalloy array and the external magnetic field strength, while inversely proportional to the size of the trap. The acoustic microstreaming activated by a piezo transducer was applied to generate fast-switching flow patterns to minimize the diffusion length scales. The flow at various driving frequencies was experimentally tested to achieve the optimal mixing effect. The flow field of the microstreaming was subsequently described by a mathematical model to understand the flow further. Finally, the prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA) were employed as model analytes to demonstrate the capability of the platform for rapid biomarker detection. With the aid of acoustic micromixing, the detection can be finished in 20 minutes. The respective limit of detection of the PSA and CEA is 0.028 ng/mL (0.8 pM) and 3.1 ng/mL (17 pM), which is respectively only 1/142 and 1/3 of the cutoff value of PSA and CEA. Our results indicate this platform has great potential for the rapid detection of multiple biomarkers in future point-of-care diagnostics.
In designing bioassay systems for low-abundance biomolecule detection, most research focuses on improving transduction mechanisms while ignoring the intrinsically fundamental limitations in solution: mass transfer and binding affinity. We demonstrate enhanced biomolecular surface binding using an acoustic nano-electromechanical system (NEMS) resonator, as an on-chip biomolecular concentrator which breaks both mass transfer and binding affinity limitations. As a result, a concentration factor of 10⁵ has been obtained for various biomolecules. The resultantly enhanced surface binding between probes on the absorption surface and analytes in solution enables us to lower the limit of detection for representative proteins. We also integrated the biomolecular concentrator into an optoelectronic bioassay platform to demonstrate delivery of proteins from buffer/serum to the absorption surface. Since the manufacture of the resonator is CMOS-compatible, we expect it to be readily applied to further analysis of biomolecular interactions in molecular diagnostics.
We have designed a pumpless acoustofluidic device for the concentration and separation of different sized particles inside a sin-gle-layered straight polydimethylsiloxane (PDMS) microfluidic channel. The proposed device comprises two parallel interdigi-tated transducers (IDTs) positioned underneath the PDMS microchannel. The IDTs produce high-frequency surface acoustic waves that generate semipermeable virtual acoustic radiation force field walls that selectively trap and concentrate larger particles at different locations inside the microchannel and allow the smaller particles to pass through the acoustic filter. The performance of the acoustofluidic device was first characterized by injecting into the microchannel a uniform flow of suspended 9.9 µm diam-eter particles with various initial concentrations (as low as 10 particles/mL) using a syringe pump. The particles were trapped with ~100% efficiency by a single IDT actuated at 73 MHz. The acoustofluidic platform was used to demonstrate the pumpless separation of 12.0 µm, 4.8 µm, and 2.1 µm microparticles by trapping the 12 µm and 4.8 µm particles using the two IDTs actuat-ed at 73 MHz and 140 MHz, respectively. However, most of the 2.1 µm particles flowed over the IDTs unaffected. The acousto-fluidic device was capable of rapidly processing a large volume of sample fluid pumped through the microchannel using an ex-ternal syringe pump. A small volume of the sample fluid was processed through the device using a capillary flow and a hydrody-namic pressure difference that did not require an external pumping device.
Current diagnosis of infectious diseases such as Hendra virus (HeV) relies mostly on laboratory
based tests. There is an urgent demand for rapid diagnosis technology to detect and identify
these diseases in humans and animals so that disease spread can be controlled. In this study,an
integrated lab-on-a-chip device using a magnetic nanoparticle immunoassay is developed. The
key features of the device are the chaotic fluid mixing, achieved by magnetically driven motion
of nanoparticles with the optimal mixing protocol developed using chaotic transport theory, and
the automatic liquid handling system for loading reagents and samples The device has been
demonstrated to detect Hendra virus antibodies in dilute horse sera samples within the short time
of 15 minutes and the limit of detection is about 0.48 ng/ml. The device platform can potentially
be used for field detection of viruses and other biological and chemical substances.
There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.
Point-of-care (POC) diagnostics is playing an increasingly important role in public health, environmental monitoring, and food safety analysis. Smartphones, alone or in conjunction with add-on devices, have shown great capability of data collection, analysis, display, and transmission, making them popular in POC diagnostics. In this article, the state-of-the-art advances in smartphone-based POC diagnostic technologies and their applications in the past few years are outlined, ranging from in vivo tests that use smartphone's built-in/external sensors to detect biological signals to in vitro tests that involves complicated biochemical reactions. Novel techniques are illustrated by a number of attractive examples, followed by a brief discussion of the smartphone's role in telemedicine. The challenges and perspectives of smartphone-based POC diagnostics are also provided.
Technologies that can enable concentration of low-abundance biomarkers are essential for early diagnosis of diseases. In this study, an optoelectrokinetic technique, termed Rapid Electrokinetic Patterning (REP), was used to enable dynamic particle manipulation in bead-based bioassays. Various manipulation capabilities, such as micro/nanoparticle aggregation, translation, sorting and patterning, were developed. The technique allows for versatile multi-parameter (voltage, light intensity and frequency) based modulation and dynamically addressable manipulation with simple device fabrication. Signal enhancement of a bead-based bioassay was demonstrated using dilute biotin-fluorescein isothiocyanate (FITC) solutions mixed with streptavidin-conjugated particles and rapidly concentrated with the technique. As compared with a conventional ELISA reader, the REP-enabled detection achieved a minimal readout of 3.87 nM, which was a 100-fold improvement in sensitivity. The multi-functional platform provides an effective measure to enhance detection levels in more bead-based bioassays.
The demand for easy to use and cost effective medical technologies inspires scientists to develop innovative lab-on-chip technologies for point-of-care in vitro diagnostic testing. To fulfill medical needs, the tests should be rapid, sensitive, quantitative, and miniaturizable, and need to integrate all steps from sample-in to result-out. Here, we review the use of magnetic particles actuated by magnetic fields to perform the different process steps that are required for integrated lab-on-chip diagnostic assays. We discuss the use of magnetic particles to mix fluids, to capture specific analytes, to concentrate analytes, to transfer analytes from one solution to another, to label analytes, to perform stringency and washing steps, and to probe biophysical properties of the analytes, distinguishing methodologies with fluid flow and without fluid flow (stationary microfluidics). Our review focuses on efforts to combine and integrate different magnetically actuated assay steps, with the vision that it will become possible in the future to realize integrated lab-on-chip biosensing assays in which all assay process steps are controlled and optimized by magnetic forces.
In part 14 of the tutorial series "Acoustofluidics--exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we provide a qualitative description of acoustic streaming and review its applications in lab-on-a-chip devices. The paper covers boundary layer driven streaming, including Schlichting and Rayleigh streaming, Eckart streaming in the bulk fluid, cavitation microstreaming and surface-acoustic-wave-driven streaming.
We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs' magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35,000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 μL) while a low limit of detection can be achieved (about 1 ng mL(-1)).
Ultrasonic radiation forces can be used for non-intrusive manipulation and concentration of suspended micrometer-sized particles. For bioanalytical purposes, standing-wave ultrasound has long been used for rapid immuno-agglutination of functionalized latex beads. More recently, detection methods based on laser-scanning fluorometry and single-step homogeneous bead-based assays show promise for fast, easy and sensitive biochemical analysis. If such methods are combined with ultrasonic enhancement, detection limits in the femtomolar region are feasible. In this paper, we review the development of standing-wave ultrasonic manipulation for bioanalysis, with special emphasis on miniaturization and ultrasensitive bead-based immunoassays.
Acoustofluidics, the fusion of acoustics and microfluidic techniques, has recently seen increased research attention across multiple disciplines due in part to its capabilities in contactless, biocompatible, and precise manipulation of micro-/nano-objects. Herein, a bimodal signal amplification platform which relies on acoustofluidics-induced enrichment of nanoparticles is introduced. The dual-function biosensor can perform sensitive immunofluorescent or surface-enhanced Raman spectroscopy (SERS) detection. The platform functions by using surface acoustic waves to concentrate nanoparticles at either the center or perimeter of a glass capillary; the concentration location is adjusted simply by varying the input frequency. The immunofluorescence assay is achieved by concentrating fluorescent analytes and functionalized nanoparticles at the center of the microchannel, thereby improving the visibility of the fluorescent output. By modifying the inner wall of the glass capillary with plasmonic Ag nanoparticle-deposited ZnO nanorod arrays and focusing analytes toward the perimeter of the microchannel, SERS sensing using the same device setup is achieved. Nanosized exosomes are used as a proof-of-concept to validate the performance of the acoustofluidic bimodal biosensor. With its sample-enrichment functionality, bimodal sensing, short processing time, and minute sample consumption, the acoustofluidic chip holds great potential for the development of lab-on-a-chip based analysis systems in many real-world applications.
The integration of acoustics and microfluidics (termed acoustofluidics) presents a frontier in the engineering of functional micro-/nanomaterials. Acoustofluidic techniques enable active and precise spatiotemporal control of matter, providing great potential for the design of advanced nanosystems with tunable material properties. In this work, we introduce an acoustofluidic approach for engineering multifunctional three-dimensional nanostructure arrays and demonstrate their potential in enrichment and biosensing applications. In particular, our acoustofluidic device integrates an acoustic transducer with a sharp-edge-based acoustofluidic reactor that enables uniform patterning of zinc oxide (ZnO) nanoarrays with customizable lengths, densities, diameters, and other properties. The resulting ZnO nanoarray-coated glass capillaries can rapidly and efficiently capture and enrich biomolecules with sizes ranging from a few nanometers to several hundred nanometers. In order to enable the detection of these biomolecules, silver (Ag) nanoparticles are deposited onto the ZnO nanoarrays, and the integrated ZnO-Ag capillary device functions as a label-free plasmonic biosensing system for surface-enhanced Raman spectroscopy (SERS) based detection of exosomes, DNA oligonucleotides, and E. Coli bacteria. The optical sensing enhancement of ZnO-Ag capillary is further validated through finite-difference time-domain (FDTD) simulations. These findings not only provide insights into the engineering of functional micro-/nanomaterials using acoustofluidics, but also shed light onto the development of portable microanalytical devices for point-of-care applications.
Ultrasound as a biocompatible and powerful approach has been advanced in biotechnology. Here we present an acoustic microchip integrating modification and detection for in-situ analysis. Such microchip employs two pairs of piezoelectric transducers (PZTs) for acoustic field generation and a polydimethylsiloxane (PDMS) microcavity on a polyethylene terephthalate (PET) substrate for producing microparticle array. The applying of acoustic field results in rapidly forming microparticle array by adjusting the inputting frequency and voltage. In-situ modification and detection are accelerated due to the dynamic ultrasonic streaming around the ultrasound induced microparticle array. Such array also benefits from reducing the detection errors by coupling of multiple points. With this strategy, biomarkers (e.g. miRNA) can be enriched, and achieve in-situ modification and detection via simple two steps with excellent specificity. After the detection, samples are regained from the output channel by releasing the acoustic field, which is benefit for further analysis. Such integrated modification and detection acoustic microchip shows great potential in visual in-situ analysis and enriching ultratrace biomarkers for clinical diagnosis.
Acoustofluidic methods, with advantages including simplicity of device design, biocompatible manipulation, and low power consumption, have been touted as promising tools for point-of-care (POC) testing. Here, we report a cell-phone-based acoustofluidic platform that uses acoustic radiation forces to enrich nanoscale analytes and red and green fluorescence nanoparticles (SiO2@R and G@SiO2) as probes for POC visual testing. Thus, the color signals from the fluorescent probes are enhanced, and colorimetric sensitivity is significantly improved. As a POC demonstration, the acoustofluidic platform is used to detect hemoglobin (Hb) from human blood, resulting in a rapid and straightforward measurement of normal blood Hb levels. Combining an acoustofluidic-based nanoparticle-concentration platform with cell-phone-based colorimetry, our method introduces a potential pathway toward practical POC testing.
Chemiluminescence immunoassay (CLIA) has been greatly developed in the past several decades due to its good sensitivity and specificity. Nowadays, CLIA has been highly improved with novel nanomaterials and newly-developed technologies. The advancement of CLIA combined with relevant technologies are reviewed in the paper, including enhanced chemiluminescent, antibody preparation, aptamer selection, nanomaterials assisted CLIA, and CLIA coupling with newly-developed technologies. Finally, some critical challenges are discussed in the field and the future development direction of CLIA is prospected. The review will be of great significance for CLIA basic research and practical applications in the fields of biomedical diagnostics, food and drug testing, environmental monitoring, and other fields.
Portable chemiluminescence (CL) imaging with a smartphone has shown a great promise for point-of-care testing of diseases, especially for acute myocardial infarction (AMI) which may occur abruptly. A challenge remains how to improve the imaging sensitivity that usually is several orders of magnitude lower than those of counterpart methodologies using the sophisticated equipment. Towards this goal, here we report the target-triggered in situ growth of [email protected] nanoprobes into spherical nucleic acid enzymes (SNAzymes), which serve as both nanolabels and amplifiers for portable CL imaging of microRNAs (miRNAs) with an ultrahigh sensitivity comparable to that of instrumental measurement under same conditions. A G-quadruplex (G4) DNA dense layer is dynamically produced on the gold nanocore via a DNAzyme machine-driven hairpin cleaving, and capture the cofactor hemin to form the SNAzymes with higher peroxidase activity and stronger nuclease-resistance than the commonly used G4 DNAzymes. The matured SNAzymes are then utilized as catalytic labels in a luminol-artesunate CL system for miRNA imaging with a smartphone as the portable detector. In this way, two AMI-related miRNAs, miRNA-499 and miRNA-133a, are successfully detected in real patients’ serum with a naked-eye visualized CL change at 10 fM, showing a five orders of magnitude improvement on the sensitivity of visualizing the same disease markers in clinical circulating blood as com-pared to the reported strategy. In addition, a good selectivity of our developed CL imaging platform is demonstrated. These unique features make it promising to employ this portable imaging platform for clinical AMI diagnosis.
Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 μm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17–1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens.
Graphical abstract
A homogeneous electrogenerated chemiluminescence (ECL) immunoassay for highly sensitive quantification of specific biomarkers based on immunomagnetic beads and homogeneous detection on a magnetic electrode was developed, for the first time. The magnetic electrode is made of a glassy carbon electrode and a series of ring permanent magnets. D-Dimer antigen was taken as a model analyte while biotinylated D-dimer antibody bound on the streptavidin-coated magnetic beads was utilized as a magnetic capture probe and ruthenium complex-labeled D-dimer antibody was employed as an ECL probe. After a fixed amount of magnetic capture probe and the ECL probe was introduced into analyte D-dimer solution, the “sandwich” immunoconjugates on the magnetic beads were formed in tested solution and then magnetically concentrated on the surface of the magnetic electrode. The homogeneous ECL immunoassay for quantification of specific biomarker was directly carried out in the presence of co-reactant tripropylamine. The low detection limit of 1 ng/mL in magnetic enrich time of 2 min and the good magnetic regeneration for the detection of D-dimer were achieved. The magnetic bead shield ECL emission was extensively discussed. This work demonstrates that the homogeneous (separation-free) ECL immunoassay using magnetic beads and magnetic electrode is a promising approach to quantify the biomarkers with high sensitivity and selectivity and in a short time. This approach can be easily extended to ECL and electrochemical biosensing for other biomarkers.
Graphical abstract
Along with the considerable potential and increasing demand of the point-of-care testing (POCT), corresponding detection platforms have attracted great interest in both academic and practical fields. The first few generations of conventional detection devices tend to be costly, complicated to operate and hard to move on account of early limitations in the level of technological development and relatively high requirement of performance. Owing to the requirements for rapidity, simplicity, accuracy and cost controlling in the POCT, reader systems are urgently needed to be developed, upgraded and modified constantly, realizing on-site testing and healthcare management without a specific place or cumbersome operation. Accordingly, numerous rapid detection platforms with diverse size and performance have emerged such as bench-top apparatuses, handheld devices and intelligent detection devices. This review discusses various devices developed mainly for the detection of lateral flow test strips (LFTSs) or microfluidic strips in the POCT and summarizes these devices by size and portability. Furthermore, on the basis of various detection methods and diverse probes usually containing specific nanoparticles composites, three most common aspects of detection rationale in the POCT are selected to elaborate each kind of detection platforms in this paper: colorimetric assay, luminescent detection and magnetic signal detection. Herein, we focus on their structures, detection mechanisms and assay results, accompany with discussions and comments on the performances, costs and potential application, as well as advantages and limitations of each technique. In addition, perspectives on the future advances of detection platforms and some conclusions are proposed.
Ionic concentration-polarization (CP)-based biomolecule preconcentration is an established method for enhancing the detection sensitivity of target biomolecules immunoassay. However, its main drawback lies in its inability to directly control the spatial overlap between the preconcentrated plug of biomolecules and the surface immobilized antibodies. To overcome this, we simultaneously preconcentrated freely suspended, surface functionalized nanoparticles and target molecules along the edge of a depletion layer, thus, increasing the binding kinetics and avoiding the need to tune their relative locations to ensure their spatial overlap. After the desired incubation time, the nanoparticles were dielectrophoretically trapped for postprocessing analysis of the binding signal. This novel combination of CP-based preconcentration and dielectrophoresis (DEP) was demonstrated through binding of avidin and biotin-conjugated particles as a model bead-based immunoassay, wherein increased detection sensitivity was demonstrated compared to an immunoassay without CP-based preconcentration. The DEP trapping of the beads following binding is important not only for enhanced detection signal due to the preconcentration of the beads at the electrode edges but also for controlling their location for future applications integrating localized sensors. In addition, DEP may be important also as a preprocessing step for controlling the number of beads participating in the immunoassay.
We intended to develop a novel biosensor using gold nanoparticles (AuNPs) for indicating different concentrations of E. coli O157:H7 and smart phone imaging APP for monitoring color change of the AuNPs. The magnetic nanoparticles (MNPs) modified with the capture antibodies and the polystyrene microspheres (PSs) modified with the detection antibodies and the catalases were simultaneously used to react with the target bacteria in the first mixing channel of the microfluidic chip, and hydrogen peroxide was injected and catalyzed by the catalases on the MNP-bacteria-PS complexes. After the mixture of the AuNPs and the crosslinking agents were injected to react with the catalysate in the second mixing channel and incubated in the detection chamber, the aggregation of the AuNPs was triggered through the crosslinking agents, resulting in the color of the AuNPs changing from blue to red. Finally, the color was measured using the smart phone imaging APP to determine the amount of the bacteria. This biosensor exhibited a good specificity and sensitivity for detection of E. coli O157:H7 in chicken samples with a lower detection limit of 50 CFU/mL.
Acoustic actuation of fluids at small scales may finally enable a comprehensive lab-on-a-chip revolution in microfluidics, overcoming long-standing difficulties in fluid and particle manipulation on-chip. In this comprehensive review, we examine the fundamentals of piezoelectricity, piezoelectric materials, and transducers; revisit the basics of acoustofluidics; and give the reader a detailed look at recent technological advances and current scientific discussions in the discipline. Recent achievements are placed in the context of classic reports for the actuation of fluid and particles via acoustic waves, both within sessile drops and closed channels. Other aspects of micro/nano acoustofluidics are examined: atomization, translation, mixing, jetting, and particle manipulation in the context of sessile drops and fluid mixing and pumping, particle manipulation, and formation of droplets in the context of closed channels, plus the most recent results at the nanoscale. These achievements will enable applications across the disciplines of chemistry, biology, medicine, energy, manufacturing, and we suspect a number of others yet unimagined. Basic design concepts and illustrative applications are highlighted in each section, with an emphasis on lab-on-a-chip applications.
We report the nonlinear acoustic streaming effect and the fast manipulation of microparticles by microelectromechanical Lamb-wave resonators in a microliter droplet. The device, consisting of four Lamb-wave resonators on a silicon die, generates cylindrical traveling waves in a liquid and efficiently drives nine horizontal vortices within a 1−μl droplet; the performance of the device coincides with the numerical model prediction. Experimentally, the particles are enriched at the stagnation center of the main vortex on the free surface of the droplet in open space without microfluidic channels. In addition, the trajectories of the particles in the droplet can be controlled by the excitation power.
We report a smartphone label-free biosensor platform based on grating-coupled surface plasmon resonance (GC-SPR). The sensor system relies on the smartphone's built-in flash light source and camera, a disposable sensor chip with Au diffraction grating and a compact disk (CD) as the spectra dispersive unit. The Au grating sensor chip was modified with a synthetic peptide receptor and employed on the GC-SPR detection of lipopolysaccharides (known as endotoxins) with detection limit of 32.5 ng/mL in water. Upon incubation of various small and macro-molecules with the synthetic peptide modified sensor chips, we concluded the good selectivity of the sensor for LPS detection. In addition, the sensor shows feasibility for the detection of LPS in commonly used clinical injectable fluids, such as clinical-grade 0.9% sodium chloride intravenous infusion, compound sodium lactate intravenous infusion and insulin aspart. The developed sensor platform offers the advantage of portability and simplicity, which is attractive for point-of-care and remote detection of biomedical and environmental targets.
In vitro biosensors have been an integral component for early diagnosis of cancer in the clinic. Among them, no-wash biosensors, which only depend on the simple mixing of the signal generating probes and the sample solution without additional washing and separation steps, have been found to be particularly attractive. The outstanding advantages of facile, convenient, and rapid response of no-wash biosensors are especially suitable for point of care testing (POCT). One fast-growing field of no-wash biosensor design involves the usage of nanomaterials as signal amplification carriers or direct signal generating elements. The analytical capacity of no-wash biosensors with respect to sensitivity or limit of detection, specificity, stability, and multiplexing detection capacity are largely improved because of their large surface area, excellent optical, electrical, catalytic, and magnetic properties. This review provides a comprehensive overview of various nanomaterial-enhanced no-wash biosensing technologies, and focuses on the analysis of the underlying mechanism of these technologies applied for the early detection of cancer biomarkers ranging from small molecules to proteins, and even whole cancerous cells. Representative examples are selected to demonstrate the proof-of-concept with promising applications for in vitro diagnostics of cancer. Finally, a brief discussion of common unresolved issues and a perspective outlook on the field are provided.
Early diagnosis of diabetic retinopathy (DR) is vital but challenging. DR is a common complication and a major cause of vision loss in patients with diabetes mellitus. Without appropriate medical intervention, visual impairment may become a great burden to our healthcare system. In clinical practice, the current diagnostic methods, such as fluorescence angiography and optical coherence tomography, remain constrained by non-quantitative examinations and individual ophthalmologists’ experiences. Late diagnosis often prevents early treatment. To address the constraints on current diagnostics, this study developed an optoelectrokinetic bead-based immunosensing technique for detecting lipocalin 1 (LCN1), a DR biomarker. The concentration level of LCN1 in the tears of DR patients increases with DR severity. The immunoassay was dependent on the formation of sandwiched immunocomplexes on the particles. A secondary antibody labeled with dyes/quantum dots (QDs) was used to visualize the presence of the target antigens. Rapid electrokinetic patterning (REP), an optoelectrokinetic technique, was used to dynamically enhance the fluorescent signal by concentrating the modified particles. The limit of detection (LOD) of the technique could reach 110 pg/mL. Only 1.5 μL of a sample fluid was required for the measurement. Our results showed that highly sensitive and improved LOD is subjected to particle stacking, small particle size, and compact cluster. By labeling different particle sizes with dyes/QDs for LCN1 and TNF-α, we successfully used REP to detect the two DR biomarkers on the same platform. The development of an optoelectrokinetic bead-based immunosensing technique can provide new insights into diagnosing other low-abundance diseases in the future.
We demonstrate a surface plasmon resonance imaging platform integrated with a smartphone to be used in the field with high-throughput biodetection. Inexpensive and disposable SPR substrates are produced by metal coating of commercial Blu-ray discs. A compact imaging apparatus is fabricated using a 3D printer which allows taking SPR measurements from more than 20.000 individual pixels. Real-time bulk refractive index change measurements yield noise equivalent refractive index changes as low as 4.12 × 10⁻⁵ RIU which is comparable with the detection performance of commercial instruments. As a demonstration of a biological assay, we have shown capture of mouse IgG antibodies by immobilized layer of rabbit anti-mouse (RAM) IgG antibody with nanomolar level limit of detection. Our approach in miniaturization of SPR biosensing in a cost-effective manner could enable realization of portable SPR measurement systems and kits for point-of-care applications.
In this work, a smartphone based optical platform for colorimetric analysis of blood hematocrit using a disposable microfluidic device is designed, implemented and fully characterized. Using an integrated camera in the smartphone, pictures of human blood in the microchannel were taken and analyzed by a mobile application. To avoid the image burning and ambient light effect, a unique light-diffusing model inside a white acrylic-imaging box was included in this platform. With the image-processing program on the smartphone, the developed device was successfully applied to determine various hematocrit levels of human blood from 10% to 65%. Furthermore, the characterization of the depth of the microfluidic channel demonstrated that a shallower depth of the microchannel enhanced the sensitivity of the hematocrit determination. The limit of detection (LOD) obtained from the developed platform was 0.1% of hematocrit with a sensitivity of 0.53 GSV (a.u.)/hematocrit%. Thus, utilizing the advantage of the microfluidic effect, a rapid and sensitive hematocrit determination was achieved successfully. With this, the hematocrit of human blood could be conveniently and accurately determined using the disposable microfluidic device, and then processed by the smartphone camera and mobile image analysis application.
The ubiquitous distribution and international connectivity of smartphones is changing the concept of mobile health and promising to reshape the biosensor market. Smartphone-based biosensors have been explored using different approaches, either using the smartphone as detector or as instrumental interface. Smartphone-based biosensors have great potential as point-of-care and point-of-need platforms for healthcare, food safety, environmental monitoring, and biosecurity, especially in remote and rural areas. Here, we critically review the most recent papers on the use of smartphones as analytical devices and biosensors. We focus on analytical performance and on prospects for commercialization.
In this manuscript, a microfluidic detection module, which allows a sensitive readout of biological assays in point-of-care (POC) tests, is presented. The proposed detection module consists of a microfluidic flow cell with an integrated Complementary Metal-Oxide-Semiconductor (CMOS)-based single photon counting optical sensor. Due to the integrated sensor-based readout, the detection module could be implemented as the core technology in stand-alone POC tests, for use in mobile or rural settings. The performance of the detection module was demonstrated in three assays: a peptide, a protein and an antibody detection assay. The antibody detection assay with readout in the detection module proved to be 7-fold more sensitive that the traditional colorimetric plate-based ELISA. The protein and peptide assay showed a lower limit of detection (LLOD) of 200fM and 460fM respectively. Results demonstrate that the sensitivity of the immunoassays is comparable with lab-based immunoassays and at least equal or better than current mainstream POC devices. This sensitive readout holds the potential to develop POC tests, which are able to detect low concentrations of biomarkers. This will broaden the diagnostic capabilities at the clinician's office and at patient's home, where currently only the less sensitive lateral flow and dipstick POC tests are implemented.
Considerable advances in point-of-care testing (POCT) devices stem from innovations in cellphone (CP)-based technologies, paper-based assays (PBAs), lab-on-a-chip (LOC) platforms, novel assay formats, and strategies for long-term reagent storage. Various commercial CP platforms have emerged to provide cost-effective mobile health care and personalized medicine. Such assay formats, as well as low-cost PBAs and LOC-based assays, are paving the way to robust, automated, simplified, and cost-effective POCT. Strategies have also been devised to stabilize reagent storage and usage at ambient temperature. Nevertheless, successful commercialization and widespread implementation of such clinically viable technologies remain subject to several challenges and pending issues.
Increasingly, smartphones are used as portable personal computers, revolutionizing communication styles and entire lifestyles. Using 3D-printing technology we have made a disposable minicartridge thatcan be easily prototyped to turn any kind of smartphone or tablet into a portable luminometer to detect chemiluminescence derived from enzyme-coupled reactions. As proof-of-principle, lactate oxidase was coupled with horseradish peroxidase for lactate determination in oral fluid and sweat. Lactate can be quantified in less than five minutes with detection limits of 0.5 mmol/L (corresponding to 4.5 mg/dL) and 0.1 mmol/L (corresponding to 0.9 mg/dL) in oral fluid and sweat, respectively. The smartphone-based device shows adequate analytical performance to offer a cost-effective alternative for non-invasive lactate measurement. It could be used to evaluate lactate variation in relation to anaerobic threshold in endurance sport and for monitoring lactic acidosis in critical-care patients.
We have developed a simple and accurate biosensor based on a chemiluminescent (CL)-lateral flow immunoassay (LFIA) method integrated in a smartphone to quantitatively detect salivary cortisol. The biosensor is based on a direct competitive immunoassay using peroxidase–cortisol conjugate, detected by adding the chemiluminescent substrate luminol/enhancer/hydrogen peroxide. The smartphone camera is used as light detector, for image acquisition and data handling via a specific application. We 3D-printed simple accessories to adapt the smartphone. The system comprises a cartridge, which houses the LFIA strip, and a smartphone adaptor with a plano-convex lens and a cartridge-insertion slot. This provides a mini-darkbox and aligned optical interface between the camera and the LFIA membrane for acquiring CL signals. The method is simple and fast, with a detection limit of 0.3 ng/mL. It provides quantitative analysis in the range of 0.3–60 ng/mL, which is adequate for detecting salivary cortisol in the clinically accepted range. It could thus find application in the growing area of home-self-diagnostic device technology for clinical biomarker monitoring, overcoming the current difficulties in achieving sensitive and quantitative information with conventional systems taking the advantage of smartphone connectivity and the enhanced performance of the included camera.
We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample, and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.
In this paper we report for the first time the use of a smartphone to image and quantify bio-chemiluminescence coupled biospecific enzymatic reactions to detect analytes in biological fluids. Using low-cost 3D printing technology we fabricated a smartphone accessory and a minicartridge for hosting biospecific reactions. As a proof-of-principle we report two assays: a bioluminescence assay for total bile acids using 3α-hydroxyl steroid dehydrogenase co-immobilized with bacterial luciferase system and a chemiluminescence assay for total cholesterol using cholesterol esterase/cholesterol oxidase coupled with the luminol-H2O2-horseradish peroxidase system. These assays can be performed within 3 min in a very straightforward manner and provided adequate analytical performance for the analysis of total cholesterol in serum (limit of detection 20 mg/dL) and total bile acid in serum and oral fluid (limit of detection 0.5 μmol/L) with a reasonable accuracy and precision. Smartphone-based bio-chemiluminescence detection could be thus applied to a variety of clinical chemistry assays.
Point-of-care testing (POCT) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application. In this review, we first describe the state of the art of medical-laboratory tests that can be performed near the patient. At present, POCT ranges from basic blood-glucose measurement to complex viscoelastic coagulation assays. POCT shortens the time to clinical decision-making about additional testing or therapy, as delays are no longer caused by transport and preparation of clinical samples, and biochemical-test results are rapidly available at the point of care. Improved medical outcome and lower costs may ensue.Recent, evolving technological advances enable the development of novel POCT instruments. We review the underlying analytical techniques. If new instruments are not yet in practical use, it is often hard to decide whether the underlying analytical principle has real advantage over former methods. However, future utilization of POCT also depends on health-care trends and new areas of application. But, even today, it can be assumed that, for certain applications, near-patient testing is a useful complement to conventional laboratory analyses.
A microfluidic system for the enrichment of biological particles, operating on the principle of dielectrophoresis (DEP), is presented. Through the use of a unique manifestation of DEP, contactless DEP (cDEP), this system illustrates the potential to sidestep the common trade-off between sample throughput and selectivity without the need of a complicated fabrication process. The ability to concentrate particles from a sample fluid is validated experimentally through the concentration of 2-μm polystyrene beads and live THP-1 human leukemia cells from a heterogeneous media solution. Finite element analysis of the electric field within the microfluidic channel of the device allows for the determination of effective experimental parameters and accurate predictions of a particle's trajectory through the device. The concentration of particles combined with a fabrication procedure conducive to mass production makes cDEP an attractive alternative to current sample enrichment technologies.
We present novel concepts to process and read out multiplexed, bead-based fluorescence immunoassays. At the start of the read-out process, a statistically arranged monolayer of color-encoded beads is aggregated in a detection chamber. Each bead is first identified by incorporated color tags which are either dyes or luminescing quantum dots (QDs). Subsequently, the reaction-specific fluorescence signal is quantified. The read-out process is accelerated by an in-house-developed image-processing algorithm. The optical read-out device consists of standard components, e.g. a color CCD-camera as detection unit, an LED as light source, optical filters, and a drive to spin the polymer disk. The liquid handling along the complete assay protocol is realized on a centrifugal lab-on-a-disk platform. We successfully demonstrate the performance of this device by the implementation of a hepatitis A and a tetanus assay.
Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 µL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 µL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.
In this study, a novel microfluidic device with microbead array was developed and sensitive genotyping of HBV was demonstrated using quantum dot as labels. This device was assembled by using two PDMS slabs featured with different microstructures and channel depths for the construction of a functional region comprising a chamber array and a single sampling microchannel. Since the chamber array and its sampling channel are of different channel depths and are bonded face-to-face, weir structures are generated to confine the microbeads which could be addressed using the microfluidic channel. Highly sensitive virus DNA detection was achieved by the enhanced mass transport in the microfluidics and the rapid reaction dynamics of suspension microbead array. The device could detect 1000 copies/mL of HBV virus in clinical serum samples using in vitro transcribed RNA as the target molecules. Based on DNA hybridization with quantum dots labels, on-chip virus genotyping was also demonstrated with high discrimination specificity and sensitivity (4 pM, S/N >3) using synthesized HBV DNA probes. This microfluidic device combines the rapid binding kinetics of homogeneous assays of microbead array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for high sensitivity virus DNA analysis with small reagent consumption, short assay time and parallel detection.
It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology.
Almost all immuno-biosensors are inherently limited by the quality of antibodies available for the target molecule, and obtaining a highly sensitive antibody for a given target molecule is a challenge. We describe a highly efficient and flexible way to enhance immunoassay detection sensitivity and binding kinetics using a nanofluidic based electrokinetic preconcentrator. The device is a microfluidic integration of charge-based biomolecule concentrator and a bead-based immunoassay. Because the preconcentrator can increase the local biomolecule concentration by many orders of magnitude, it gives the immuno-sensor better sensitivity and faster binding kinetics. With a 30 min preconcentration, we were able to enhance the immunoassay sensitivity (with molecular background) by more than 500 fold from higher 50 pM to the sub 100 fM range. Moreover, by adjusting the preconcentration time, we can switch the detection range of the given bead-based assay (from 10-10 000 ng ml(-1) to 0.01-10 000 ng ml(-1)) to have a broader dynamic range of detection. As the system can enhance both detection sensitivity and dynamic range, it can be used to address the most critical detection issues in the detection of common disease biomarkers.