Fluorescent Immunoassays for Detection and Quantification of Cardiac Troponin I: A Short Review.

Cardiovascular diseases are considered one of the major causes of human death globally. Myocardial infarction (MI), characterized by a diminished flow of blood to the heart, presents the highest rate of morbidity and mortality among all other cardiovascular diseases. These fatal effects have triggered the need for early diagnosis of appropriate biomarkers so that countermeasures can be taken. Cardiac troponin, the central key element of muscle regulation and contraction, is the most specific biomarker for cardiac injury and is considered the "gold standard". Due to its high specificity, the measurement of cardiac troponin levels has become the predominant indicator of MI. Various forms of diagnostic methods have been developed so far, including chemiluminescence, fluorescence immunoassay, enzyme-linked immunosorbent assay, surface plasmon resonance, electrical detection, and colorimetric protein assays. However, fluorescence-based immunoassays are considered fast, accurate and most sensitive of all in the determination of cardiac troponins post-MI. This review represents the strategies, methods and levels of detection involved in the reported fluorescence-based immunoassays for the detection of cardiac troponin I.

Discrepancy between Cardiac Troponin Assays Due to Endogenous Antibodies.

BACKGROUND: Despite well-described analytical effects of autoantibodies against cardiac troponin (cTn) I on experimental assays, no study has systematically examined their impact on cTn assays in clinical use. We determined the effects of endogenous antibodies on 5 different cTnI assays and a cTnT assay. METHODS: cTn was measured by 6 methods: Siemens hs-cTnI Centaur, Siemens hs-cTnI Vista, Abbott hs-cTnI Architect, Beckman hs-cTnI Access, Beckman cTnI Access, and Roche hs-cTnT Elecsys. Measurements were repeated on 5 assays (all except Siemens hs-cTnI Vista) following immunoglobulin depletion by incubation with protein A. Low recovery of cTnI (<40%) following immunoglobulin depletion was considered positive for macro-cTnI. Protein A findings were validated by gel filtration chromatography and polyethylene glycol precipitation. RESULTS: In a sample of 223 specimens selected from a community laboratory that uses the Siemens hs-cTnI Centaur assay and from which cTn was requested, 76% of samples demonstrated increased cTnI (median, 88 ng/L; interquartile range, 62-204 ng/L). Macro-cTnI was observed in 123 (55%) of the 223 specimens. Comparisons of cTnI assays markedly improved once patients with macro-cTnI were removed. Passing-Bablok regression analysis between hs-cTnI assays demonstrated different slopes for patients with and without macro-cTnI. In patients with macro-cTnI, 89 (72%) showed no effect on the recovery of cTnT, whereas 34 (28%) had reduced recovery of cTnT. The proportion of results above the manufacturers' 99th percentile varied with the cTn assay and macro-cTnI status. CONCLUSION: We suggest that the observed discrepancy between hs-cTnI assays may be attributed in part to the presence of macro-cTnI.

Automated, Universal, and Mass-Producible Paper-Based Lateral Flow Biosensing Platform for High-Performance Point-of-Care Testing.

Paper-based lateral flow assays (LFAs) are among the most widely used biosensing platforms for point-of-care testing (POCT). However, the conventional colloidal gold label of LFAs show low sensitivity and limited quantitative capacity. Alternatively, the use of enzyme/chemical reaction-based signal amplification with structural modifications has enhanced analytical capacity but requires multiple user interventions as a trade-off, increasing complexity, test imprecision, and time. These platforms are also difficult to manufacture, limiting their practical applications. In this study, within the current LFA production framework, we developed a highly sensitive, automated, universal, and manufacturable LFA biosensing platform by (i) incorporating gold nanoparticles into a polymer-networked peroxidase with an antibody as a new scheme for enhanced enzyme conjugation and (ii) integrating a mass-producible and time-programmable amplification part based on a water-swellable polymer for automating the sequential reactions in the immunoassay and signal amplification, without compromising performance, simplicity, and production feasibility. We applied this platform to evaluate cardiac troponin I (cTnI), a gold-standard biomarker for myocardial infarction diagnosis. Quantitative analysis of cTnI in clinical setting remains limited to the laboratory-based high-end and costly standard equipment. Coupled with an enzyme-catalyzed chemiluminescence method, this platform enables automated, cost-effective (0.66 USD per test), and high-performance testing of human cTnI in serum samples within 20 min with a detection range of 6 orders of magnitude, detection limit of 0.84 pg mL-1 (595-fold higher than conventional cTnI-LFA), and a coefficient of variation of 2.9-8.5%, which are comparable to the standard equipment and acceptable for clinical use. Moreover, cTnI analysis results using clinical serum/plasma samples revealed a strong correlation (R2 = 0.991) with contemporary standard equipment, demonstrating the practical application of this platform for high-performance POCT.

Simple one-pot aqueous synthesis of 3D superstructured PtCoCuPd alloyed tripods with hierarchical branches for ultrasensitive immunoassay of cardiac troponin I.

By integrating the amplified electrochemical signals and effective capture of antibodies together, advanced multimetallic superstructured nanocrystals endow label-free immunosensors promising applications in early diagnosis and monitoring of diseases. Herein, four-metallic PtCoCuPd hierarchical branch-like tripods (HBTPs) were directly synthesized by a green one-pot aqueous method without any seed or organic solvent involved, which were applied to construct a novel label-free immunosensor for detecting cardiac troponin I (cTnI). The specific hierarchical micro/nanostructures greatly improved the immobilization of antibodies and enhanced the catalytic activity for K3Fe(CN)6, which would effectively amplify the electrochemical signals, thereby improving the detection sensitivity. Under the optimal conditions, the as-developed immunosensor exhibited a wide linear range (0.001-100.0 ng mL-1) and a low detection limit (0.2 pg mL-1, S/N = 3) for the assay of cTnI. The immunosensor provides a powerful platform for quantitative detection of cTnI, which can be explored to detect other tumor markers in actual sample analysis.

Novel electrochemical immunosensor for sensitive monitoring of cardiac troponin I using antigen-response cargo released from mesoporous Fe3O4.

A novel controlled release system-based antigen-response electrochemical immunosensor was developed for detecting cardiac troponin I (cTnI) by using aminated polystyrene microsphere (APSM) as molecular gate and Fe3O4 as nanocontainer. The amino functionalized mesoporous Fe3O4 (Fe3O4-NH2) was used to load cobalt phthalocyanine nanoparticles (CoPc NPs) and further capture the antibody of cTnI (Ab) to form Fe3O4-Ab. In addition, APSM was introduced to cap on the mesoporous of Fe3O4-Ab by electrostatic interaction. With the addition of cTnI, APSM was separated from Fe3O4-Ab due to the specific binding of antibody to antigen. Then, CoPc NPs were released from the mesoporous. The experimental results revealed that CoPc NPs showed superb catalytic performance when catalyzing hydrogen peroxide (H2O2) reduction in phosphate buffer saline (PBS). The current responses are correlated with the amount of cTnI. Under the best conditions, a broad linear range from 1.0 pg/mL to 100 ng/mL with a low detection limit of 0.39 pg/mL (S/N = 3) was obtained. The immunosensor also shows good reproducibility and selectivity, which endows its broad application prospect in clinical research.

Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration.

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

Thionin functionalized signal amplification label derived dual-mode electrochemical immunoassay for sensitive detection of cardiac troponin I.

A sensitive sandwich-type electrochemical immunosensor was established by employing Au@Pt core-shell multi-branched nanoparticles, and thionin functionalized nitrogen/sulfur co-doped graphene oxide (N/S-cGO/L-lys/Au@Pt MBs/Thi) as a double signal label to detect cardiac troponin I (cTnI). In this work, Au nanorods functionalized polydopamine (Au NR@PDA) with high adsorption capacity and superior electroconductivity can provide an efficient substrate for immobilizing primary antibodies (Ab1). In the proposed N/S-cGO/L-lys/Au@Pt MBs/Thi, an electrochemically active molecule, Thi was covalently bonded in the N/S-cGO/L-lys/Au@Pt MBs. It presented a strong differential pulse voltammetry (DPV) current signal without electron transfer mediators, and showed a high electrocatalytic activity toward H2O2 reduction by using amperometric i-t (i-t). Impressively, with the synergistic effect of N/S-cGO/L-lys/Au@Pt MBs/Thi and Au NR@PDA, the developed dual-mode electrochemical immunosensor for cTnI detection showed a wide linear concentration range (50 fg/mL to 250 ng/mL, 750 fg/mL to 100 ng/mL) and a low detection limit (16.7 fg/mL, 250 fg/mL) via i-t and DPV, respectively. Furthermore, this immunosensor exhibited acceptable reproducibility, high sensitivity and good stability under optimal conditions. More importantly, the satisfactory results were obtained in detection of cTnI-spiked human serum samples, and the presented method may be a promising application in clinical bioanalysis.

Facile and clean synthesis of dihydroxylatopillar[5]arene-stabilized gold nanoparticles integrated Pd/MnO2 nanocomposites for robust and ultrasensitive detection of cardiac troponin I.

In this work, a novel, facile, clean synthesis of monodisperse Au nanoparticles (AuNPs) with an average diameter of 5 nm was achieved by reducing HAuCl4 with dihydroxylatopillar[5]arene (2HP5) in basic solution without the use of harsh reagents and/or external energy. Accordingly, toluidine blue (TB), one electrochemcial indicator, could enter into the cavity of 2HP5 to fabricate host-guest complex through strong electrostatic interaction and charge-transfer interaction, which significantly enhanced the loading quantity of TB and effectively suppressed the leaking of TB resulting in an ultrasensitive and robust electrochemical response. More importantly, the integration of 2HP5-stabilized AuNPs and Pd-decorated MnO2 nanocomposites (2HP5@Au-Pd/MnO2) might usually obtain a novel functional-enhanced materials and lead to new properties and improving the analytical performance and robustness of electrochemical devices. Therefore, we construct a sandwich-type electrochemical immunosensor using TB-2HP5@Au-Pd/MnO2 nanocomposites as the transducing materials for robust and ultrasensitive detection of cardiac troponin I (cTnI), a significant biomarker of acute myocardial infarction. As anticipated, this immunosensor had remarkable robustness, sensitivity, stability, specificity, and corresponded linearly to the concentration of cTnI over a wide range from 0.005 to 20 ng mL-1 with a low detection limit of 2 pg mL-1 (S/N = 3). The proposed electrochemical immunosensor showed acceptable recoveries in human serum, indicating that macrocycle-stabilized metal nanoparticle might be a promising emerging transducer material for the detection of biological markers.

Paper based modification-free photoelectrochemical sensing platform with single-crystalline aloe like TiO2 as electron transporting material for cTnI detection.

By controlling target-induced signal quencher release, a label-free and modification-free microfluidic paper based photoelectrochemical analytical device (µ-PAD) for cardiac troponin-I (cTnI) detection was designed for the first time. To achieve it, cellulose paper based single-crystalline three-dimensional aloe like TiO2 arrays (PSATs) were firstly fabricated as the electron transporting material, providing direct pathways for the charge carriers transfer, and subsequently coupled with CdS to form PSATs/CdS heterojunction for extending the solar spectrum response. Meanwhile, positive charged mesoporous silica nanoparticles (PMSNs) were prepared as the nanocarrier to efficient entrap the Cu2+ which could be regarded as signal quencher due to their reaction with CdS to form CuxS. Single stranded DNAs (ssDNAs), which could bind specifically with the target of cTnI, were then introduced to couple with the PMSNs and used as the bio-gate to encapsulate the signal quencher of Cu2+, endowing the functional PMSNs with responsiveness to cTnI. When the cTnI existed, the ssDNAs were dissociated from PMSNs due to the formation of cTnI-ssDNAs complexes, triggering controllable release of the trapped Cu2+, and further decreasing the photocurrent signal caused by the formation of CuxS. Accordingly, the concentration of cTnI could be accurately quantified via the photocurrent, realizing the target-induced modification-free µ-PAD assay. We believe this work could provide an ingenious idea to construct the easy-to-use novel modification-free µ-PAD.

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I.

BACKGROUND: Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135-209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146-158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1-37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. METHODS: Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1-77 and cTnI135-209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. RESULTS: cTnI135-209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. CONCLUSIONS: Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. GENERAL SIGNIFICANCE: cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.

Simple, low-cost, sensitive and label-free aptasensor for the detection of cardiac troponin I based on a gold nanoparticles modified titanium foil.

This research demonstrated the electrochemical modification of low-cost titanium (Ti) metal substrate with gold nanoparticles (AuNPs) for the aptamer-based detection of cardiac troponin I (cTnI). AuNPs were deposited onto Ti sheets by the potential-step deposition method with high density and homogeneity as well as good crystallinity. It was then applied as a transducer to immobilize a thiol-functionalized DNA aptamer via the self-assembled monolayer mechanism for the specific binding of cTnI. This was verified through electrochemical and morphological analyses. The aptasensor could detect cTnI in a linear range of 1-1100 pM with a detection limit of ca. 0.18 pM. The aptasensor showed high sensitivity and specificity to cTnI over other interfering compounds with good recoveries in the diluted human serum samples.

Development of cardiac troponin-I biosensor based on boron nitride quantum dots including molecularly imprinted polymer.

The cardiac Troponin-I (cTnI) is one of the subunits of cardiac troponin complexes and a pivotal biochemical marker of acute myocardial infarction (AMI). Due to its myocardial specificity, cTnI is widely used for the diagnosis of AMI diseases. In this study, a novel imprinted biosensor approach based on boron nitride quantum dots (BNQDs) was presented for cTnI detection in plasma samples. Various characterization methods such as scanning electron microscope (SEM), transmission electron microscope (TEM), x-ray diffraction (XRD), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for all characterizations of nanomaterials. After the characterization analysis, cTnI imprinted electrode was developed in the presence of 100.0 mM pyrrole containing 25.0 mM cTnI. After that, the analytical studies of cTnI in plasma samples were performed by using cTnI imprinted biosensor. The results of the study have revealed that 0.01-5.00 ng mL-1 and 0.0005 ng mL-1 were found as the linearity range and the detection limit (LOD). Moreover, the selectivity of cTnI imprinted glassy carbon electrode (GCE) was investigated for plasma sample analysis in the presence of other nonspecific and specific proteins including cardiac myoglobin (MYG), bovine serum albumin (BSA) and cardiac troponin T (cTnT), respectively. Furthermore, the prepared biosensor was examined in terms of stability, repeatability, reproducibility and reusability. Finally, the imprinted biosensor was applied to the plasma samples having high recovery.

A Multi-Region Magnetoimpedance-Based Bio-Analytical System for Ultrasensitive Simultaneous Determination of Cardiac Biomarkers Myoglobin and C-Reactive Protein.

Cardiac biomarkers (CBs) are substances that appear in the blood when the heart is damaged or stressed. Measurements of the level of CBs can be used in course of diagnostics or monitoring the state of the health of group risk persons. A multi-region bio-analytical system (MRBAS) based on magnetoimpedance (MI) changes was proposed for ultrasensitive simultaneous detection of CBs myoglobin (Mb) and C-reactive protein (CRP). The microfluidic device was designed and developed using standard microfabrication techniques for their usage in different regions, which were pre-modified with specific antibody for specified detection. Mb and CRP antigens labels attached to commercial Dynabeads with selected concentrations were trapped in different detection regions. The MI response of the triple sensitive element was carefully evaluated in initial state and in the presence of biomarkers. The results showed that the MI-based bio-sensing system had high selectivity and sensitivity for detection of CBs. Compared with the control region, ultrasensitive detections of CRP and Mb were accomplished with the detection limits of 1.0 pg/mL and 0.1 pg/mL, respectively. The linear detection range contained low concentration detection area and high concentration detection area, which were 1 pg/mL⁻10 ng/mL, 10⁻100 ng/mL for CRP, and 0.1 pg/mL⁻1 ng/mL, 1 n/mL⁻80 ng/mL for Mb. The measurement technique presented here provides a new methodology for multi-target biomolecules rapid testing.

Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in lateral flow assay with core-shell SERS nanotags.

Rapid and sensitive quantification of multiplex proteins in a wide concentration range is challenging in high throughput analysis. Herein, we proposed a lateral flow assay (LFA) based on core-shell surface enhanced Raman scattering (SERS) nanotags for multiplex and quantitative detection of cardiac biomarkers for the early diagnosis of acute myocardial infarction (AMI). In practice, Raman dyes (RDs) were embedded into the interior-gap of silver core and gold shell nanoparticles (NPs) to form SERS nanotags as labels instead of gold colloids and three test lines were employed in the strip for the detection of three cardiac biomarkers, Myo, cTnI, and CK-MB, respectively. Due to the amplified signal of the SERS nanotags and the high surface area to volume ratio (SVR) of porous nitrocellulose (NC) membrane, ultrasensitive quantification of protein markers with wide linear dynamic range (LDR) was realized, which is crucial for the quick detection of multiplex biomarkers in the same sample without pretreatments at bedsides. This method makes it possible for LFA in point of care testing (POCT) to be comparable with chemiluminescence immunoassay (CLIA) used in labs.

A novel label-free photoelectrochemical sensor based on N,S-GQDs and CdS co-sensitized hierarchical Zn2SnO4 cube for detection of cardiac troponin I.

A novel label-free photoelectrochemical (PEC) sensor based on graphene quantum dots doped with nitrogen and sulfur (N,S-GQDs) and CdS co-sensitized hierarchical Zn2SnO4 cube was fabricated to detect cardiac troponin I (cTnI). The unique hierarchical Zn2SnO4 cube was synthesized successfully by the solvothermal method, which has a large specific surface to load functional materials. N,S-GQDs nanoparticles were assembled to the surface of cubic Zn2SnO4 coated ITO electrode, which efficiently accelerated the electronic transition and improved photo-to-current conversion efficiency. Then, CdS nanoparticles further were modified by in-situ growth method to form Zn2SnO4/N,S-GQDs/CdS composite with prominent photocurrent, which was 30 times that of the Zn2SnO4 cube alone. In this work, the specific immune recognition between cTnI antigens and cTnI antibodies (anti-cTnI) reduced the intensity of the photoelectric signal. And the intensity decreased linearly with the logarithm of cTnI concentration range from 0.001 ng/mL to 50 ng/mL with a detection limit of 0.3 pg/mL. With high sensitivity, excellent selectivity, good stability and reproducibility, the fabricated PEC sensor showed promising applications in the sensor, clinical diagnosis of myocardial infarction and PEC analysis.

Ultrasensitive label-free optical microfiber coupler biosensor for detection of cardiac troponin I based on interference turning point effect.

Sensitive detection of cardiac biomarkers is critical for clinical diagnostics of myocardial infarction (MI) while such detection is quite challenging due to the ultra-low concentration of cardiac biomarkers. In this work, a label-free immunosensor based on optical microfiber coupler (OMC) has been developed for the ultrasensitive detection of cardiac troponin I (cTnI), a selective and highly sensitive biomarker of acute myocardial infarction (AMI). CTnI monoclonal antibodies were immobilized on the surface of the fiber through polyelectrolyte layer using layer-by-layer deposition technique. For refractive index sensing characterization, an ultra-high sensitivity of 91777.9 nm/RIU was achieved when the OMC works around the dispersion turning point, which is the highest experimental demonstration in the field of fiber-optic evanescent biosensors. For biosensing, the immunosensor with good specificity showed a linear wavelength shift in the range of 2-10 fg/mL and an ultra-low detection limit of 2 fg/mL. Such immunosensors have huge application potential for the detection of cardiac biomarkers of myocardial infarction due to simple detection scheme, quick response time, ease of handling and miniaturation.

Novel peptidylated surfaces for interference-free electrochemical detection of cardiac troponin I.

Novel peptidylated surfaces were designed to minimise interferences when electrochemically detecting cardiac troponin I in complex biological samples. Disulfide-cored peptide dendrons featuring carbomethoxy groups were self-assembled on gold electrodes. The carbomethoxy groups were deprotected to obtain carboxylic groups used to immobilise antibodies for cardiac troponin I marker. The chemisorption of two types of peptides, one containing triazole and the other with native peptide bonds, on a gold substrate was studied by quartz crystal microbalance (QCM), surface plasmon resonance (SPR) and X-ray photoelectron spectroscopy (XPS). Peptides formed ordered self-assembled monolayers, contributing to a more efficient display of the subsequently immobilised antibodies towards their binding to the antigen. As a result, electrochemical immunosensors prepared by self-assembly of peptides afforded higher sensitivities for cardiac troponin I than those prepared by the chemisorption of alkane thiolated compounds. Triazolic peptide-modified immunosensors showed extraordinary sensitivity towards cardiac troponin I [1.7µA/(ng/mL) in phosphate buffer], but suffered from surface fouling in 10% serum. Modification with non-triazolic peptides gave rise to anti-fouling properties and still enabled the detection of cardiac troponin I at pg/mL concentrations in 10% serum without significant matrix effects.

Overcoming difficulties on synthesis of cardiac troponin-I.

Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.