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Mixing during Trapping Enabled a Continuous-Flow Microfluidic Smartphone Immunoassay Using Acoustic Streaming

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Mixing during Trapping Enabled a Continuous-Flow Microfluidic Smartphone Immunoassay Using Acoustic Streaming

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... 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]. ...
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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.
... 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. ...
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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.
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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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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
Article
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
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.