Sensitive Detection Of Protein Biomarkers Research Articles

Bionic E-Eye Based High-Throughput Immunocolorimetric Sensor System for Sensitive Detection BNP in Blood through Carbon-Gold Nanocomposites

Introduction Precise and sensitive detection of protein biomarkers is of vital importance in clinical diagnosis and biomedical research. To date, enzyme-linked immunosorbent assay (ELISA) remains the gold standard in detecting protein biomarkers due to the robust properties of simple operation, acceptable sensitivity, and automated high throughput [1, 2]. Despite desirable results from ELSA, pursuing high sensitivity and accuracy to meet diagnosis demands is still an ongoing endeavor. For that, nanomaterials behaving with favorable physical properties and biocompatibility may serve as a solution, especially the carbon nanosphere (CN) or gold nanoparticles (AuNPs) that feature strong stability, easy manufacture, low cost, and environment-friendly, and abundant active sites [3, 4].Heart failure (HF) is one of the most threatening cardiovascular diseases. B-type natriuretic peptide (BNP), a vital cardiac biomarker, has been acknowledged as one of the principal biomarkers for HF [5]. Individuals at risk of HF demonstrate raised BNP in serum and real-time quantification of HF biomarkers, identifying those at highest risk of HF early, and ensuring they receive appropriate treatment can effectively prevent premature death [6]. Although current ELISA-methods for detecting BNP have been developed, there are urgent needs for providing more efficiently and reliably in vitro diagnostic (IVD) methods or point-of-care (POC) devices in BNP clinical measurement.Most of the previous studies for ELISA-based BNP detection required large instruments with high costs such as microplate readers, spectrophotometers. In this work, we adopted a self-developed portable bionic electronic eye (Bionic E-eye) system using a smartphone (iPhone 4S and iPad 3) integrated with simple accessories including a piece of electroluminescent, a wide-angle lens, and a dark hood instead of the commercial microplate reader to make the entire analysis process more intelligent and convenient. In addition, the CN-AuNPs nanocomposites (CGN) we prepared was used as a signal amplifier to develop a high-throughput immunocolorimetric method that sensitively identified BNP through the color change of antibody-antigen reactions using enzyme-substrate and horseradish peroxidase-antibody (pAb-HRP) linked CGN immunoprobe (CGNs@ pAb-HRP). The combination of Bionic E-eye and high-throughput immunocolorimetric system aimed to establish a general and reliable strategy for POC testing (Figure. 1A). Method Monoclonal antibody against BNP were immobilized on the surface of the 96-well plate as capture antibody. Then the different concentrations of BNP were added to the wells. Finally, the HRP-labeled polyclonal BNP (pAb-HRP) antibody or the pAb-HRP modified CGNs (pAb-HRP-CGNs) were added to bind with BNP. After 3,3',5,5'-Tetramethylbenzidine (TMB) and HCl were added, different shades of yellow color related to BNP concentration appeared. Combining with the Bionic E-eye, BNP concentration could be analyzed by image processing. Results and Conclusions To achieve optimally sensitive signal output and fabricate a well-performance immunoprobe, the synthesized conditions were optimized. Under optimal conditions, we have successfully synthesized stable and homogeneous CGNs validated by TEM (Figure. 1B) and XPS. The performance for BNP detection by a commercial microplate reader (Figure. 1C) and Bionic E-eye were evaluated and compared. The detection range of both was comparable (7.8 ppb~125 ppb) while the Bionic E-eye system had a lower limit of detection (LOD) than a commercial microplate reader (4.3 ppb < 7.2 ppb) (Figure. 1D), indicating that compared with a commercial colorimetric reader the self-developed system achieved higher sensitivity which could be attributed to the superiority of saturation channel in HSV or RGB color model the Bionic e-Eye used. In addition, the signal amplifying effect of the pAb-HRP-CGNs probe was also confirmed in the comparison of LOD and detection range with pAb-HRP. Figure. 1E showed that the LOD in the method using pAb-HRP-CGNs reached 0.92 ppb and the wider detection range of 3.9 ppb~250 ppb was achieved compared with that of 7.8 ppb~125 ppb in the non-CGNs method. Based on the above results, this proposed CGNs based detection of BNP with Bionic e-Eye shows promising application prospect but more experimental optimizations should be performed to further improve its performance.

A Peptide-PNA Hybrid Beacon for Sensitive Detection of Protein Biomarkers in Biological Fluids.

Specific and rapid detection of proteins in biological fluids poses a challenging problem. In biological fluids, many proteins are present at low concentrations, requiring high affinity and specificity of the beacon-protein interaction. We report the design of a peptide-PNA hybrid beacon that exploits the dimeric nature of a target protein, S100B, a biomarker for brain trauma, to enhance binding affinity and specificity. The complementary base-pairing of the PNA bases brings the two arms of the beacon, one carrying an Alexa tag and the other carrying a Dabcyl moiety, into proximity, thus quenching Alexa fluorescence. Each of the arms carries a sequence that binds to one of the subunits. Binding to the target separates the quencher from the probe lifting the quenching of fluorescence. Enhanced affinity and specificity resulting from simultaneously binding to two sites allowed specific detection of S100B at low-nanomolar concentrations in the presence of serum. The design can be easily adapted for the detection of proteins containing multiple binding sites and could prove useful for rapid and sensitive biomarker detection.

Surface optimization of gold nanoparticle mass tags for the sensitive detection of protein biomarkers via immuno-capture LI-MS.

HPV-induced cervical cancer is one of the most lethal cancers. Therefore, the development of a reliable and accurate method for the early diagnosis of HPV infections is highly important. Here, gold nanoparticles (AuNPs) were utilized as mass tags in an immuno-capture LI-MS assay for the detection of HPV marker proteins. Through the optimization of the amount of antibodies and surface charges on AuNPs, high antigen detection efficiency with minimal non-specific binding was achieved. With optimized antibody-conjugated AuNPs, low attomole amount of HPV proteins in HeLa cell lysate was quantified.

A ternary probe for target-triggered autonomous multi-branch rolling circle amplification for highly sensitive colorimetric sensing of platelet-derived growth factor BB

Highly sensitive detection of protein biomarkers has an important role in clinical diagnosis and disease treatment. On the basis of a newly designed ternary probe containing the aptamer, primer and circular DNA sequences, we have established an ultrasensitive and label-free colorimetric approach for detecting platelet-derived growth factor BB protein biomarkers via a target-triggered autonomous multi-branch rolling circle amplification (mbRCA) strategy. The target molecules specifically bind the aptamer sequences in the ternary probes and causes the configuration transition of the primers to trigger mbRCA generation of numerous of G-quadruplex sequences with the presence of the assistance hairpin. Hemin further associates with these G-quadruplexes to yield many hemin/G-quadruplex DNAzymes with the horseradish peroxidase-mimicking function, which catalyze the substrate solution to induce intense color transition for ultrasensitive and label-free colorimetric determination of the target molecules with a 0.055 pg mL−1 of detection limit in the range of 0.1–1000 pg mL−1. This aptamer-based sensing method exhibits high selectivity and can achieve the detection of low levels of protein biomarkers in diluted serum samples, which offers the developed approach new opportunities for detecting other trace protein biomarkers for the purpose of early disease diagnosis.

Dual Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich Assay for the Specific and Sensitive Detection of Protein Biomarkers.

Molecularly imprinted polymers (MIPs), which are synthesized in the presence of a template, have been widely used as antibody mimics for important applications. Through the combination with a highly sensitive detection scheme such as chemiluminescence and surface-enhanced Raman scattering (SERS), MIP-based sandwich assays have emerged as promising analytical tools for the detection of disease biomarkers. However, so far, MIPs have been used only as target-capturing probes, whereas labeling by other means was needed, which limits the application range. Herein, we present a new approach, called a dual MIP-based plasmonic immunosandwich assay (duMIP-PISA), for the specific and sensitive detection of protein biomarkers in complex biological samples. A C-terminal epitope-imprinted self-assembled gold nanoparticle monolayer-coated glass slide was prepared as a plasmonic substrate for the specific extraction of target protein, while N-terminal epitope-imprinted Raman-responsive Ag@SiO2 nanoparticles were prepared as nanotags for the specific labeling of captured protein. The formed MIP-protein-MIP sandwich-like complexes could produce a significantly enhanced SERS signal. The dual MIP-based recognitions ensured high specificity of the assay, while SERS detection provided ultrahigh sensitivity. The duMIP-PISA of neuron-specific enolase (NSE) in human serums was demonstrated, which permitted the differentiation of small cell lung cancer patients from healthy individuals. As compared to regular ELISA, the duMIP-PISA exhibited multiple merits including a simpler procedure, faster speed, lower sample volume requirement, and wider linear range. The approach well demonstrated the great potentials of MIPs and can be easily modified and extended to other protein biomarkers. Therefore, the duMIP-PISA approach holds great promise in many important applications such as disease diagnosis.

Highly Sensitive Detection of Protein Biomarkers Using Graphene Field-Effect Transistor Integrated with a Microfluidic Platform

In this work, we present a highly sensitive biosensing platform for the detection of protein biomarkers by utilizing aptamer-modified graphene field-effect transistors (GFETs) contained within a microfluidic chip. We have chosen thrombin as a model biomarker and its corresponding DNA-based aptamer as a target receptor which is anchored to the graphene surface to modulate the drain current of the GFET. The proposed biosensing platform has the potential to be used in early-stage disease diagnosis and in applications related to lab-on-a-chip-based health monitoring technology. Aptamer-based biosensing offers many advantages including low-cost, chemical and thermal stability, mass producibility, long shelf-life and reusability. Among different target analytes, protein biomarker detection is critical in early screening and diagnostics of diseases. Thrombin is an important protein biomarker which is produced by the body and plays a significant role in blood clotting or coagulation process or regulation of tumor growth [1]. Therefore, detection of thrombin is key to monitoring a number of cardiovascular diseases. Graphene-based FET (GFET) has become a highly promising candidate as next-generation biosensors due to the various inherent advantages it offers such as facile integration with the existing planar technology due to its 2-dimensional structure, label-free sensing, and high sensitivity. However, one of the major challenges with GFET-based biosensing is the lack of reproducibility due to each graphene layer having different physical dimensions and therefore electronic properties. Moreover, since GFET is highly sensitive to the charge disturbances at the FET channel, the sample liquid that is in contact with the graphene surface must be controlled. Therefore, integration of GFET biosensor with a microfluidic device is expected to enhance the sensing performances in terms of sensitivity and reproducibility. In this work, we have implemented a microfluidic chip integrated with GFET device to build a miniaturized platform for aptamer-based detection of a protein biomarker. The GFET devices were fabricated by transferring CVD (chemical vapor deposition) grown graphene onto the microfabricated gold electrodes on the Si/SiO2substrate where the electrodes form the source and the drainas schematically depicted in Figure 1. The FET devices were then sealed with a PDMS block containing a microfluidic channel as shown in Figure 2. The graphene channel was then functionalized with amine reactive 1-pyrene butyric acid N-hydroxysuccinimide ester (PBASE) by delivering the PBASE dissolved in dimethyl formamide through the microfluidic channel. The PBASE was immobilized on graphene surface via the π-π stacking interaction between graphene and the pyrene group of PBASE. The graphene channel was then further functionalized with the aptamers by flowing the amine terminated thrombin-binding aptamer solution through the microfluidic chip. The aptamers were crosslinked with the PBASE on graphene surface via the covalent linkage between the amine-reactive succinimide group of PBASE and the amine group of the aptamers [2]. For the biomarker detection, various concentrations of thrombin solutions were introduced to the GFET device through the microfluidic flow system. For a given concentration of thrombin, the drain current (IDS) vs. gate voltage (VGS) characteristics were measured in order to observe the shift in the Dirac voltage (VDirac). Figure 3 shows the Raman spectra of graphene on Si/SiO2 substrate before and after PBASE functionalization. The additional peak at 1618 cm-1 in Figure 3(a) represents the pyrene group resonance peak due to π – π stacking interaction [3] between the aromatic pyrenyl group of PBASE and the basal plane of graphene, confirming the immobilization of PBASE on graphene. Figure 4 shows the UV-vis spectra of the device sample before and after the aptamer immobilization. The peak at 260 nm indicates the presence of aptamers on the device surface. The resulting measurements are presented in Figure 5. It can be seen that introduction of thrombin causes the Dirac voltage to shift to the right suggesting p-doping of the graphene surface [4]. This is consistent with the cationic nature of thrombin at neutral pH. The corresponding calibration curve is shown in Figure 6. The sensor shows a linear behavior over a wide range of concentrations from 1 nM to 1µM.

Series of In Situ Photoinduced Polymer Graftings for Sensitive Detection of Protein Biomarkers via Cascade Amplification of Liquid Crystal Signals.

Developing of new polymeric materials for the sensitive and rapid detection of trace protein biomarkers has attracted increasing attention in biomedical fields. Herein, series of in situ photoinduced polymer graftings were developed for sensitive detection of protein biomarkers by using featured cascade amplification of liquid crystal (LC) signals. The limit-of-detection (LOD) for native bovine serum albumin (BSA) molecules is around 10 μg/mL in a LC biosensor before signal amplification. Upon the cascade amplification using surface-grafted polymers, poly[poly(ethylene glycol) methacrylate] grafting ( s-P(PEGMA)) exhibits superior amplification ability (104-fold lower than native BSA) than the other two graftings of poly(2-hydroxyethyl methacrylate) ( s-PHEMA) and poly(methacrylic acid) ( s-PMAA; 102-fold lower than native BSA). The contact angles of water and LC on the s-P(PEGMA) grafting show significant difference in comparison with s-PHEMA and s-PMAA graftings ( p < 0.05), implying interfacial energies of the grafted polymers may dictate the orientational transition of LCs. The clinical urine samples collected from the patients with proteinuria were also used to confirm the feasibility of the polymer-amplified LC sensors for practical protein assays. The present work reveals that in situ photoinduced polymer grafting is one promising method to amplify the signals of LC biosensors for the rapid and sensitive detection of trace protein biomarkers.

A target-responsive autonomous aptamer machine biosensor for enzyme-free and sensitive detection of protein biomarkers.

Highly sensitive and selective detection of protein disease markers is crucial for fundamental research and disease diagnosis. In this work, based on a new target-triggered autonomous aptamer machinery amplification approach, we developed a simple and sensitive sensing platform for the detection of a cancer biomarker, platelet-derived growth factor BB (PDGF-BB), in human sera. The target PDGF-BB binds the fluorescently quenched hairpin aptamer probes and causes structural and conformational changes in the aptamers to recover their fluorescence. The presence of partial duplex DNA fuel strands further leads to the recycling of the target proteins and the functioning of the aptamer machine autonomously to amplify the fluorescence emission significantly, thereby resulting in sensitive detection of PDGF-BB down to the low picomolar level. Besides, due to the high specificity of the aptamer, such a protein detection method shows high selectivity and can be employed to detect PDGF-BB in human sera. Featuring the advantages of being enzyme-free and nanomaterial-free for signal amplification, as well as homogeneous detection, the developed approach thus holds great potential for the construction of various aptasensors for the convenient and sensitive detection of different molecules.

Highly Sensitive Detection of Protein Biomarkers with Organic Electrochemical Transistors.

The analysis of protein biomarkers is of great importance in the diagnosis of diseases. Although many convenient and low-cost electrochemical approaches have been extensively investigated, they are not sensitive enough in the detection of protein biomarkers with low concentrations in physiological environments. Here, this study reports a novel organic-electrochemical-transistor-based biosensor that can successfully detect cancer protein biomarkers with ultrahigh sensitivity. The devices are operated by detecting electrochemical activity on gate electrodes, which is dependent on the concentrations of proteins labeled with catalytic nanoprobes. The protein sensors can specifically detect a cancer biomarker, human epidermal growth factor receptor 2, down to the concentration of 10-14 g mL-1 , which is several orders of magnitude lower than the detection limits of previously reported electrochemical approaches. Moreover, the devices can successfully differentiate breast cancer cells from normal cells at various concentrations. The ultrahigh sensitivity of the protein sensors is attributed to the inherent amplification function of the organic electrochemical transistors. This work paves a way for developing highly sensitive and low-cost biosensors for the detection of various protein biomarkers in clinical analysis in the future.

Immuno-PCR with digital readout

Immuno-PCR (IPCR) combines the versatile ELISA antigen detection with ultrasensitive PCR signal amplification, thereby enabling the highly sensitive detection of a broad range of targets with a typically very large dynamic detection range. The quantification of the antigen is usually achieved by real-time PCR, which provides a correlation between the target concentration and amplified DNA marker. We here report on the implementation of digital droplet PCR as a means for direct quantification of DNA copies to enable the highly sensitive detection of protein biomarkers. To this end, two alternative approaches, based on either magnetic microbead-based IPCR or a microplate-release IPCR were tested. The latter format worked well and revealed an extraordinary high robustness and sensitivity. While rtIPCR already fulfills typical immunoassay acceptance criteria, ddIPCR enables improved accuracy and precision of the assay because signal response and analyte concentrations are directly correlated. The utility of the novel ddIPCR technology is demonstrated at the example of two cytokines, interleukin 2 and interleukin 6 (IL2, IL6, respectively), with an overall average CV% of 5.0 (IL2) and 7.4 (IL6).

Surface Enhanced Laser Desorption Ionization of Phospholipids on Gold Nanoparticles for Mass Spectrometric Immunoassay.

High-throughput and sensitive detection of proteins are essential for clinical diagnostics and biomarker discovery. We develop a novel high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay method, which utilizes antibody-modified phospholipid bilayer coated gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized magnetic beads as the capture reagent. This method enables magnetic enrichment of the PBL-AuNPs label specific to target protein, allowing sensitive surface enhanced laser desorption ionization (SELDI)-TOF MS detection of the protein via its specific label. AuNPs act as not only the support but also the matrix for the phospholipids in SELDI TOF MS detection. Moreover, with phospholipids with varying molecular weights as the encoded MS reporters, this method allows multiplexed detection of multiple proteins. With the use of a predefined phospholipids internal standard, this method also affords excellent reproducibility in protein quantification. We have demonstrated this method using the assays of two tumor biomarkers, and the results reveal that it provides a sensitive platform for multiplexed protein detection with detection limits in the picomolar ranges. This method may provide a useful platform for high-throughput and sensitive detection of protein biomarkers for clinical diagnostics.

Highly sensitive detection of protein biomarkers via nuclear magnetic resonance biosensor with magnetically engineered nanoferrite particles.

Magnetic-based biosensors are attractive for on-site detection of biomarkers due to the low magnetic susceptibility of biological samples. Here, we report a highly sensitive magnetic-based biosensing system that is composed of a miniaturized nuclear magnetic resonance (NMR) device and magnetically engineered nanoferrite particles (NFPs). The sensing performance, also identified as the transverse relaxation (R2) rate, of the NMR device is directly related to the magnetic properties of the NFPs. Therefore, we developed magnetically engineered NFPs (MnMg-NFP) and used them as NMR agents to exhibit a significantly improved R2 rate. The magnetization of the MnMg-NFPs was increased by controlling the Mn and Mg cation concentration and distribution during the synthesis process. This modification of the Mn and Mg cation directly contributed to improving the R2 rate. The miniaturized NMR system, combined with the magnetically engineered MnMg-NFPs, successfully detected a small amount of infectious influenza A H1N1 nucleoprotein with high sensitivity and stability.

Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serums

Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5pM–10.0nM and a detection limit of 0.12pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.

Urine reflection of changes in blood.

The most important of nature of biomarker is changes. Blood is under strict homeostatic control which means changes tend to be removed from blood. Urine is a partial filtrate of blood, reflects systemic physiology but with no homeostatic mechanism. However, changes induced directly into the blood can be more sensitively detected in urine than in blood itself. This indicates that urine may serve as a source for more sensitive detection of protein biomarkers than blood.

Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma.

The most fundamental property of biomarkers is change. But changes are hard to maintain in plasma since it is strictly controlled by homeostatic mechanisms of the body. There is no homeostatic mechanism for urine. Besides, urine is partly a filtration of blood, and systematic information can be reflected in urine. We hypothesize that change of blood can be reflected in urine more sensitively. Here we introduce the interference into the blood by two anticoagulants heparin or argatroban. Plasma and urine proteins were profiled by LC-MS/MS and then validated by Western blot in totally six SD female rats before and after the drug treatments. In argatroban treated group, with exactly the same experimental procedure and the same cutoff value for both plasma and urine proteins, 62 proteins changed in urine, only one of which changed in plasma. In heparin treated group, 27 proteins changed in urine but only three other proteins changed in plasma. Both LC-MS/MS and Western blot analyses demonstrated drug-induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.

Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma (591.2)

Biomarker studies commonly monitor the composition of plasma, which is under strict homeostatic control, while urine is not. Urine is a partial filtrate of blood; and its composition reflects systemic physiology. Here we show that changes in the coagulation status of blood can be more sensitively detected in urine than in plasma. Plasma and urine protein composition was analysed by LC‐MS/MS and Western blot in six SD female rats before and after treatment with heparin or argatroban. LC‐MS/MS and Western blot analyses demonstrated drug‐induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.Grant Funding Source: Supported by the National Basic Research Program of China

Rapid and Sensitive Detection of Nano-fluidically Trapped Protein Biomarkers

ABSTRACTIn this work, we demonstrate the label-free and ultrasensitive detection of troponin-T, cardiac biomarker using nanoporous membrane integrated on a microelectrode sensor platform. The nanoporous membrane allows for spatial confinement of the protein molecules. Antigen interaction with thiol immobilized antibody perturbs the electrical double layer. Charge perturbations are recorded as impedance change at low frequency using the principle of electrochemical impedance spectroscopy (EIS). The measured impedance change is used to quantitatively determine the concentration of troponin-T in tested sample. We have shown that sensitivity of sensor for troponin-T to be 1pg/mL. The accuracy and reliability of this sensor was tested by comparing the experimental troponin-T concentration values with a commercially available electrochemiluminescence assay measured with Roche Elecsys analyzer. Using this technique we were successful in detecting protein biomarkers in whole blood, human serum, and ionic buffers. This technology provides a robust analytical platform for rapid and sensitive detection of protein biomarkers, thus establishing this technology as an ideal candidate for biomarker screening in clinical settings.

Nanoparticles for Enhanced Sensitivity in Electrochemical Immunoassays

In this manuscript, we report on electrochemical biosensors based on various nanoparticles (NPs) as labels for sensitive detection of protein biomarkers. We used silica nanoparticle as a carrier to loading a large amount of electroactive species such as poly(guanine) for sensitive immunoassay of tumor necrosis factor-alpha (TNF-α). We took the advantages of the unique hollow structure and reconstruction properties of apoferritin to prepare Cd3(PO4)2 nanoparticles as labels for sensitive assay of TNF-α. A novel immunochromatographic/electro-chemical biosensor based on quantum dots as labels has also been developed for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. These biosensors are quite sensitive with the detection limit at pM level and these approaches based on nanoparticle labels offer a new avenue for sensitive detection of protein biomarkers.

Nanoparticles Based Electrochemical Immunoassay for Sensitive Detection of Protein Biomarkers

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