Detection Of Human Serum Proteins Research Articles

A diagnostic electrochemical aptasensor development for sCD80 protein detection in human serum

Elevating soluble CD80 (sCD80) in human serum is a natural response to autoimmune diseases such as rheumatoid arthritis (RA). The level of sCD80 is associated with RA development and prognosis; therefore, it is potentially used as a biomarker. sCD80 is commonly measured in human serum using immunoassays (e.g., ELISA) with multiple drawbacks, mainly cross-reactivity. Aptamer-based biosensors (aptasensors) development for quantifying and detecting different biological molecules demonstrates applicability in next-generation medicine and biomarker detection. Herein, we selected a specific aptamer for sCD80 by conventional in-vitro selection process (SELEX) with the high-affinity aptamer (Kd = 47.69 nM). A sensitive aptasensor, for the first time, was developed on a screen-printed gold electrode (AuSPE) platform compatible with easy-to-use label-free electrochemical impedance spectroscopy. The immobilization of the aptamer on the gold surface and the presence of sCD80 in a complex with the aptamer were characterized by photo-induced force microscopy, which revealed the uniform assembly of the aptamer monolayer and the distribution of sCD80 on the electrode surface. The developed aptasensor showed a linear performance (0.025–10.0 nM of protein) with a detection limit of 8.0 pM. Furthermore, the aptasensor was tested in a biological matrix, where a linear signal was observed for the increased amount of spiked sCD80 (R2 = 0.9887). The recovery of the spiked amounts ranged from 105 to 125% with coefficient of variation (CV%) <7%, which supported the applicability of this sensor in detecting sCD80 for diagnosis.

A graphdiyne-based protein molecularly imprinted biosensor for highly sensitive human C-reactive protein detection in human serum

• A graphdiyne-based protein molecularly imprinted biosensor was developed. • The biosensor achieved a highly sensitive detection towards C-reactive protein. • Graphdiyne with high conductivity and biocompatibility was utilized for biosensor. • Developed biosensor achieved target recognition in complex biological samples. • The biosensor exhibited excellent antifouling capability. Traditional protein molecularly imprinted polymers (MIPs) are used to develop biosensors for highly specific protein recognition based on generated three-dimensional cavities that are completely complementary to the imprinted template molecules. As a new generation of artificial identification probes, protein MIPs can realize the recognition specificity, but the analytical sensitivity and limit of detection (LOD) are not optimal. Here, a highly sensitive protein MIPs biosensor was constructed, in which conductive and biocompatible graphdiyne (GDY) nanosheets, antifouling and specific MIPs were combined, to achieve the highly sensitive and selective recognition of human C-reactive protein. Dopamine, owing to its simple polymerization procedure and the verified low fouling property of polydopamine, was used as the functional monomer to form steady complex with the template molecule through hydrogen bonding and multipoint electrostatic attraction. GDY with high biocompatibility and conductivity was independently synthesized and initially introduced into the C reactive protein imprinted polymers (C-MIPs) to enhance the electrochemical response and provide a suitable microenvironment for the bioactive protein molecules. In addition to the highly specific MIPs, antifouling material was also utilized to further improve the recognition selectivity. Herein, our developed C-MIPs biosensor performed with a wider linear detection range from 10 −5 to 10 3 ng/mL and the LOD was found to be 0.41 × 10 −5 ng/mL. Moreover, the C-MIPs biosensor showed excellent selectivity, reversibility and reusability, a short rebinding time, and long-term stability. Notably, the developed C-MIPs biosensor performed well even in complex serum samples without discernible signal suppression, indicating its good antifouling ability, as well as high sensitivity and selectivity. The developed C-MIPs biosensor behaved well in human blood samples, declaring its potential practical application capability.

Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates.

Simple SummaryTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is the most lethal infectious disease from a single pathogen for which there is no effective vaccine available. Rhesus macaques are extremely susceptible to MTB and therefore represent relevant models to study the pathogenesis of TB and assess the potential of TB drugs and vaccines. However, there are no diagnostic tools currently available that allow rapid, user-friendly detection of TB for either TB research purposes or monitoring nonhuman primate colonies. To develop a rapid diagnostic test, we investigated whether low complexity lateral flow assays (LFAs) that we recently developed for rapid and quantitative detection of human serum proteins are applicable to detect and monitor active pulmonary TB in NHPs. We found that serum levels of SAA1, IP-10, and IL-6 detected by LFAs were significantly increased after MTB infection in rhesus macaques. Moreover, levels of these biomarkers correlated with disease severity as determined by pathology scoring and allowed detection of the effect of vaccination and drug treatment in experimentally MTB infected macaques. These UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be used to assess new therapeutic and prophylactic treatment methods in macaques to tackle TB.Nonhuman primates (NHPs) are relevant models to study the pathogenesis of tuberculosis (TB) and evaluate the potential of TB therapies, but rapid tools allowing diagnosis of active pulmonary TB in NHPs are lacking. This study investigates whether low complexity lateral flow assays utilizing upconverting reporter particles (UCP-LFAs) developed for rapid detection of human serum proteins can be applied to detect and monitor active pulmonary TB in NHPs. UCP-LFAs were used to assess serum proteins levels and changes in relation to the MTB challenge dosage, lung pathology, treatment, and disease outcome in experimentally MTB-infected macaques. Serum levels of SAA1, IP-10, and IL-6 showed a significant increase after MTB infection in rhesus macaques and correlated with disease severity as determined by pathology scoring. Moreover, these biomarkers could sensitively detect the reduction of bacterial levels in the lungs of macaques due to BCG vaccination or drug treatment. Quantitative measurements by rapid UCP-LFAs specific for SAA1, IP-10, and IL-6 in serum can be utilized to detect active progressive pulmonary TB in macaques. The UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be applied in studies on TB vaccine and drug development involving macaques.

Fabrication of an electrochemical biosensor composed of multi-functional Ag ion intercalated DNA four-way junctions/rhodium nanoplate heterolayer on a micro-gap for C-reactive protein detection in human serum.

As inflammation plays a role in the pathogenesis of acute coronary syndromes, C-reactive protein (CRP) can be used as a biomarker. To detect CRP precisely, the authors prepared a CRP electrochemical biosensor consisting of an eight Ag ion-intercalated multifunctional DNA four-way junction (MF-DNA-4WJ) and a porous rhodium nanoparticle (pRhNP) heterolayer on a micro-gap electrode. To increase conductivity, we used eight Ag+ ion-inserted DNA four-way junctions through a C-C mismatch. Each DNA 4WJ was designed to have the CRP aptamer sequence, an anchoring region (thiol group), and two of four C-C mismatch regions at the end of the fragments. After an annealing step, the MF-DNA-4WJ assembly configuration and selective binding of CRP were confirmed through native TBM-PAGE (Tris-borate-magnesium chloride-polyacrylamide gel electrophoresis). The Au micro-gap electrode was fabricated to load 5 μl of the sample, and this was performed during eight experiments on one chip to establish the accuracy of the data. Then, pRhNPs were immobilized on a Au micro-gap electrode using cysteamine. To confirm the electrochemical properties, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were conducted. The durability of pRhNPs was confirmed through CV. To test the sensing performance of the prepared CRP biosensor, the limit of detection (LOD) and selectivity tests were conducted using EIS. The results indicated that charge transfer resistance (Rct) can be used efficiently to probe these interactions within the variable CRP concentration range, from 1 pM to 100 nM (0.23 ng L-1-23 μg L-1). The LOD of this sensor was 0.349 pM (0.08 ng L-1) (at S/N = 3). As a result of diluting the CRP to the same concentration range in a 20% human serum sample, the LOD was 3.55 fM (0.814 pg L-1) (at S/N = 3).

Photoluminescent lateral flow based on non-radiative energy transfer for protein detection in human serum

A new paper-based lateral flow immunoassay configuration was engineered and investigated. The assay is intended for the detection of a model protein in human serum, that is, human immunoglobulin G, with the aim to demonstrate a virtually universal protein detection platform. Once the sample is added in the strip, the analyte is selectively captured by antibody-decorated silica beads (Ab-SiO2) onto the conjugate pad and the sample flows by capillarity throughout the strip until reaching the test line, where a sandwich-like immunocomplex takes place due to the presence of antibody-functionalized QDs (Ab-QDs) onto the test line. Eventually, GO is added as a revealing agent and the photoluminescence of those sites protected by the complex Ab-SiO2/Antigen/Ab-QDs will not be quenched, whereas those photoluminescent sites directly exposed are expected to be quenched by GO, including the control line, made of bare QDs, reporting that the assay occurred successfully. Hence, the photoluminescence of the test line is modulated by the formation of sandwich-like immunocomplexes. The proposed device achieves a limit of detection (LOD) of 1.35ngmL−1 in standard buffer, which is lower when compared with conventional lateral flow technology reported by gold nanoparticles, including other amplification strategies. Moreover, the resulting device was proven useful in human serum analysis, achieving a LOD of 6.30ngmL−1 in this complex matrix. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications, and can be easily transferred to other analytes such as clinically relevant protein biomarkers.

Target-induced catalytic hairpin assembly formation of functional Y-junction DNA structures for label-free and sensitive electrochemical detection of human serum proteins

The construction of simple and highly sensitive aptasensors will significantly advance the monitoring of proteins. In this work, we have developed a convenient and label-free protein-induced catalytic hairpin assembly signal amplification approach for aptamer-based sensitive electrochemical detection of thrombin in human serums. The target thrombin binds the aptamer-containing hairpin probes immobilized on the sensor electrodes and triggers catalytic assembly of other two hairpins to form many G-quadruplex Y-junction DNA structures in situ. These G-quadruplex structures further associate with hemin to form G-quadruplex/hemin complexes, which are then subject to electrochemical measurement and generate substantially amplified current output for label-free and sensitive detection of thrombin. The developed aptasensor shows a dynamic range of 0.01–1.0nM and a detection limit of 6.0p.m for thrombin detection. The sensor can also discriminate the target thrombin against other non-target proteins and exhibits promising results for detecting thrombin in human serums. With the successful construction of the thrombin aptasensor, our signal amplified method can be potentially applied to detect other target molecules by choosing appropriate aptamer/ligand pairs.

An ultrasensitive label-free immunoassay for C-reactive protein detection in human serum based on electron transfer

A rapid, ultrasensitive, and practical label-free electrochemical immunoassay for measuring C-reactive protein (CRP) in real serum samples was developed.

A highly sensitive “turn‐on” fluorescent sensor for the detection of human serum proteins based on the size exclusion of the polyacrylamide gel

A highly sensitive “turn‐on” fluorescent sensor for the detection of human serum proteins based on the size exclusion of the polyacrylamide gel

A highly sensitive “turn‐on” fluorescent sensor for the detection of human serum proteins based on the size exclusion of the polyacrylamide gel

A highly sensitive "turn-on" fluorescent sensor based on the size exclusion of the polyacrylamide gel was developed for the on-gels detection of human serum proteins after PAGE. The possible mechanism of this fluorescence sensor was illustrated and validated by utilizing five kinds of colloidal silver nanoparticles with different particle size distribution and six kinds of polyacrylamide gels with different pore size. It was attributed to that silver nanoparticles (<5 nm in diameter) had been selectively absorbed into the gel and formed the small silver nanoclusters, resulting in the red fluorescence. Using this new technique for the detection of human serum proteins after PAGE, a satisfactory sensitivity was achieved and some relatively low-abundance proteins (e.g. zinc-alpha-2-glycoprotein), which are the significant proteinic markers of certain diseases can be easily detected, but not with traditional methods. Furthermore, it was also successfully applied to distinguish between serums from hepatoma patient and healthy people. As a new protein detection technique, the colloidal silver nanoparticles based "turn-on" fluorescent sensor offers a rapid, economic, low background, and sensitive way for direct detection of human serum proteins, showing available potential and significance in the development of nanobiotechnology and proteome research.

Application of Carbon Quantum Dots in Fluorescent Imaging of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A new method of carbon quantum dots (CQDs) fluorescent imaging for human serum proteins detection after polyacrylamide gels electrophoresis (PAGE) is established. Polyacrylamide gel electrophoresis is one of the most general and powerful technique to separate complex biosamples, and it is widely used in molecular biology, biochemistry and medicine. With the evolution of clinical proteomics, the development of a novel method to detect serum proteins after PAGE with high resolution and high sensitivity is of great significant. As far as we know, the carbon quantum dots have not been applied in the detection of serum proteins after PAGE. The fluorescent carbon quantum dots were synthesized by a one-step microwave pyrolysis method: glycerol and phosphate buffer (7.1 mmolL -1 , pH 7.4) (φ=70%) was mixed evenly, and then put the solu- tion into the advanced microwave digestion system and heated for 14 min (750 W), the colorless solution turned to yellow after reaction. The yellow solution was diluted into the incubation solution with HOAc-NaOAc buffer (80 mmolL -1 , pH 2.7), and then used for staining serum proteins. The emission and excitation spectra of carbon quantum dots were measured, and the excitation wavelength allowed the use of an ultraviolet lamp at 365 nm for the fluorescent imaging. In order to inves- tigate the performance of CQDs fluorescent imaging, the dilution of human serum samples separated by electrophoresis was used for detection, and was compared with traditional method of CBB-R250 staining and silver staining. The results sug- gested that CQDs based on fluorescent imaging could effectively detect human serum proteins with high sensitivity and resolution. The sensitivity of CQDs imaging was higher than CBB staining and comparable to silver staining. Therefore, the CQDs fluorescent imaging could be an inexpensive, time-saving, pollution-free and convenient method with high sensitivity and resolution for the detection of human serum proteins. It demonstrates the CQDs fluorescent imaging technology has great significant prospects in the development of biotechnology and nanotechnology. Keywords carbon quantum dot; polyacrylamide gel electrophoresis; protein; fluorescent imaging

The application of Au nanoclusters in the fluorescence imaging of human serum proteins after native PAGE: Enhancing detection by low-temperature plasma treatment

Proteins in human serum are increasingly being studied for their roles in a wide variety of biochemical interactions. To improve the sensitivity of the detection of human serum proteins after native polyacrylamide gel electrophoresis (PAGE), we have developed a fluorescence imaging detection technique for the detection. BSA (bovine serum albumin)-stabilized Au nanoclusters (NCs) were applied as fluorescent probes for imaging, and low-temperature plasma (LTP) treatment of the Au NCs was introduced to enhance the fluorescence imaging. Here, a series of optimization experiments (e.g. those to optimize for pH) were conducted for protein detection after 1-DE and 2-DE, and several types of discharge gases (He, O2, and N2) were selected for the LTP treatment. The possible mechanism of interaction between the proteins and the Au NCs was demonstrated by an isothermal titration calorimetry experiment. Using the present method, a sensitivity of 7–14 times higher than that of traditional staining detection methods was observed in the oxygen LTP-treated Au NCs fluorescence images, and some relatively low abundance proteins (identified by the MS/MS technique) were easily detected. In addition, this fluorescence imaging method was applied to distinguish between the serum samples of patients with liver diseases and those of healthy people. Thus, this fluorescence imaging method is suitable for the highly sensitive detection of various serum proteins, and it shows potential capabilities for clinical diagnosis.

The Application of Amine‐Terminated Silicon Quantum Dots on the Imaging of Human Serum Proteins after Polyacrylamide Gel Electrophoresis (PAGE)

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si QDs, were optimized and the possible mechanisms of Si QDs-protein interaction were analyzed. The mode of Si QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of α-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si QDs, but not with CBB-R250 staining. The Si QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis.

Novel Application of Ag Nanoclusters in Fluorescent Imaging of Human Serum Proteins after Native Polyacrylamide Gel Electrophoresis (PAGE)

We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry.

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.

Designing immunodipstick assay for rapid detection of human serum proteins based on mesoporous silica and colloidal gold nanoparticls

Designing immunodipstick assay for rapid detection of human serum proteins based on mesoporous silica and colloidal gold nanoparticls

Direct CdTe Quantum‐Dot‐Based Fluorescence Imaging of Human Serum Proteins

Direct detection of human serum proteins after gel electrophoresis is achieved by a CdTe quantum dot (QD)-based fluorescence imaging technique. Compared with staining methods, it offers higher sensitivity and resolution, and is resistant to photobleaching and photo- or chemical degradation. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Novel Application of Carbon Nanotubes for Improving Resolution in Detecting Human Serum Proteins with Native Polyacrylamide Gel Electrophoresis

This paper describes a novel application of carbon nanotubes for improving the resolution of a native PAGE in the detection of human serum proteins. Carbon nanotubes were functionalized and introduced into the gel of native PAGE system, and the electropherogram showed sharp, clear bands. Furthermore, the separation of some most important proteins was improved, and the established method could be applied for the detection of sera from patients with liver diseases.

Copper(II)−Alizarin Red S Complex as an Efficient Chemiluminescent Probe for the Detection of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry.

A novel probe for chemiluminescent image detection of proteins in two‐dimensional gel electrophoresis

Carrier ampholytes were found to enhance the chemiluminescence (CL) emission from the 3-aminophthalic hydrazide (luminol)-hydrogen peroxide system. They can be used as a chemiluminescent probe for rapid detection of major proteins in gels. This probe attracted much interest due to its ability to attach proteins, and to the possibility to combine it with separation techniques generating the CL emission directly. Increased signal intensity was achieved employing optimized concentrations of the carrier ampholyte enhancer. The binding of carrier ampholyte to proteins was found to occur at the pI of the proteins. Proteins from different regions of the gels were identified by their matrix-assisted TOF mass spectra and by appropriate database search, the results illustrating the possibility of major protein detection in human serum. Direct CL image detection with the carrier ampholyte probe can be applied for the detection of characteristic proteins in patients, i.e., proteins which cannot be detected without the probe.

Direct chemiluminescent imaging detection of human serum proteins in two‐dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.