A simple and fast method to study the hydrodynamic size difference of protein disulfide isomerase in oxidized and reduced form using gold nanoparticles and dynamic light scattering.

The hydrodynamic dimension of a protein is a reflection of both its molecular weight and its tertiary structures. Studying the hydrodynamic dimensions of proteins in solutions can help elucidate the structural properties of proteins. Here we report a simple and fast method to measure the hydrodyamic size of a relatively small protein, protein disulfide isomerase (PDI), using gold nanoparticle probes combined with dynamic light scattering. Proteins can readily adsorb to citrate-capped gold nanoparticles to form a protein corona. By measuring the average diameter of the gold nanoparticles before and after protein corona formation, the hydrodynamic diameter of the protein can be deduced from the net particle size increase of the assay solution. This study found that when the disulfide bonds in PDI are reduced to thiols, the reduced PDI exhibits a smaller hydrodynamic diameter than the oxided PDI. This finding is in good agreement with the X-ray diffraction analysis of PDI in single crystals. In comparison with other techniques that are used for protein hydrodynamic size analysis, the current method is easy to use, requires a trace amount of protein samples, with results obtained in minutes instead of hours.

[Diagnosis value of combined detection of serum golgi protein 73, desgamma carboxy prothrombin and α-fetoprotein in primary hepatic carcinoma].

OBJECTIVE: To analyze the clinical value of the detection of serum desgamma carboxy prothrombin (DCP), golgi glycoprotein 73 (GP73), heat-shock protein 70 (HSP70) in primary hepatic carcinoma (PHC) diagnosis. METHODS: Enzyme-1inked immunosorbent assay and electrochemiluminescence immunoassay were used to detect the serum DCP, GP73, HSP70 and α-fetoprotein (AFP) levels in 35 PHC patients and 35 healthy controls. RESULTS: AFP, DCP and GP73 levels in PHC patients were 13.780 (1.140-8 487.000)µg/L, 3 213.953 (2.510-53 994.602)pg/ml and 76.838 (24.500-232.875)ng/ml respectively, significantly higher than those in healthy controls (1.240 (0.605-5.310)µg/L, 104.610 (0.000-4 138.770)pg/ml and 30.770 (16.343-87.453)ng/ml, U value were 134.50, 258.00 and 168.00, all P < 0.01); HSP70 could not be detected in any objects. The area under the ROC curve of DCP or GP73 was 0.789, 0.863 respectively. The sensitivity and specificity to PHC diagnosis were 54.3% and 97.1% by DCP (2 939.4 pg/ml as cut-off value), 85.7% and 74.3% by GP73 (41.3 ng/ml as cut-off value), 45.7% and 100% by AFP (20 µg/L as cut-off value). In combined detection the sensitivity, specificity and accuracy were 88.6%, 74.3%, 81.4% by GP73/AFP and 91.4%, 71.4%, 81.4% by GP73/DCP/AFP. CONCLUSION: GP73, DCP are new effective markers in the diagnosis of PHC, and the combined detection of GP73, DCP and AFP can improve the diagnosis value of PHC.

Predicting detection limits of enzyme-linked immunosorbent assay (ELISA) and bioanalytical techniques in general.

The detection limit is one of the most important performance parameters for bioanalytical techniques. Here we present a generic method to estimate the detection limit of biomolecular assays based on a step-by-step analysis of the assay procedure. Enzyme-linked immunosorbent assay (ELISA) is used here as an example; however, much of the information presented in this article may be applied to other types of biomolecular assays and analytical techniques. A clear understanding of what affects the detection limit can help researchers to evaluate different bio-analytical techniques properly, and to design better strategies to optimize and achieve the best analytical performance.

Gold nanoparticle-enabled biological and chemical detection and analysis.

Gold nanoparticles (AuNPs) are some of the most extensively studied nanomaterials. Because of their unique optical, chemical, electrical, and catalytic properties, AuNPs have attracted enormous amount of interest for applications in biological and chemical detection and analysis. The purpose of this critical review is to provide the readers with an update on the recent developments in the field of AuNPs for sensing applications based on their optical properties. An overview of the optical properties of AuNPs is presented first, followed by a more detailed literature survey. As the last part of this review, we compare the advantages and disadvantages of each technique, briefly discuss their commercialization status, and some technical issues that remain to be solved in order to move the technique forward (151 references).

Developing a nanoparticle test for prostate cancer scoring.

BACKGROUND: Over-diagnosis and treatment of prostate cancer has been a major problem in prostate cancer care and management. Currently the most relevant prognostic factor to predict a patient's risk of death due to prostate cancer is the Gleason score of the biopsied tissue samples. However, pathological analysis is subjective, and the Gleason score is only a qualitative estimate of the cancer malignancy. Molecular biomarkers and diagnostic tests that can accurately predict prostate tumor aggressiveness are rather limited. METHOD: We report here for the first time the development of a nanoparticle test that not only can distinguish prostate cancer from normal and benign conditions, but also has the potential to predict the aggressiveness of prostate cancer quantitatively. To conduct the test, a prostate tissue lysate sample is spiked into a blood serum or human IgG solution and the spiked sample is incubated with a citrate-protected gold nanoparticle solution. IgG is known to adsorb to citrate-protected gold nanoparticles to form a "protein corona" on the nanoparticle surface. From this study, we discovered that certain tumor-specific molecules can interact with IgG and change the adsorption behavior of IgG to the gold nanoparticles. This change is reflected in the nanoparticle size of the assay solution and detected by a dynamic light scattering technique. Assay data were analyzed by one-way ANOVA for multiple variant analysis, and using the Student t-test or nonparametric Mann-Whitney U-tests for pairwise analyses. RESULTS: An inverse, quantitative correlation of the average nanoparticle size of the assay solution with tumor status and histological diagnostic grading was observed from the nanoparticle test. IgG solutions spiked with prostate tumor tissue exhibit significantly smaller nanoparticle size than the solutions spiked with normal and benign tissues. The higher grade the tumor is, the smaller the nanoparticle size is. The test particularly revealed large differences among the intermediate Grade 2 tumors, and suggested the need to treat them differently. CONCLUSION: Development of a new nanoparticle test may provide a quantitative measure of the prostate cancer aggressiveness. If validated in a larger study of patients with prostate cancer, this test could become a new diagnostic tool in conjunction with Gleason Score pathology diagnostics to better distinguish aggressive cancer from indolent tumor.

A facile nanoparticle immunoassay for cancer biomarker discovery.

BACKGROUND: Gold nanoparticles (AuNPs) scatter light intensely at or near their surface plasmon wavelength region. Using AuNPs coupled with dynamic light scattering (DLS) detection, we developed a facile nanoparticle immunoassay for serum protein biomarker detection and analysis. A serum sample was first mixed with a citrate-protected AuNP solution. Proteins from the serum were adsorbed to the AuNPs to form a protein corona on the nanoparticle surface. An antibody solution was then added to the assay solution to analyze the target proteins of interest that are present in the protein corona. The protein corona formation and the subsequent binding of antibody to the target proteins in the protein corona were detected by DLS. RESULTS: Using this simple assay, we discovered multiple molecular aberrations associated with prostate cancer from both mice and human blood serum samples. From the mice serum study, we observed difference in the size of the protein corona and mouse IgG level between different mice groups (i.e., mice with aggressive or less aggressive prostate cancer, and normal healthy controls). Furthermore, it was found from both the mice model and the human serum sample study that the level of vascular endothelial growth factor (VEGF, a protein that is associated with tumor angiogenesis) adsorbed to the AuNPs is decreased in cancer samples compared to non-cancerous or less malignant cancer samples. CONCLUSION: The molecular aberrations observed from this study may become new biomarkers for prostate cancer detection. The nanoparticle immunoassay reported here can be used as a convenient and general tool to screen and analyze serum proteins and to discover new biomarkers associated with cancer and other human diseases.

A 1-minute blood test detects decreased immune function and increased clinical risk in COVID-19 patients.

Upon infection with SARS-CoV-2, the virus that causes COVID-19, most people will develop no or mild symptoms. However, a small percentage of the population will become severely ill, and some will succumb to death. The clinical severity of COVID-19 has a close connection to the dysregulation of the patient's immune functions. We previously developed a simple, nanoparticle-enabled blood test that can determine the humoral immune status in animals. In this study, we applied this new test to analyze the immune function in relation to disease severity in COVID-19 patients. From the testing of 153 COVID-19 patient samples and 142 negative controls, we detected a drastic decrease of humoral immunity in COVID-19 patients who developed moderate to severe symptoms, but not in patients with no or mild symptoms. The new test may be potentially used to monitor the immunity change and predict the clinical risk of patients with COVID-19.

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study.

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 microg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.

Poly(acrylic acid)-stabilized colloidal gold nanoparticles: synthesis and properties.

Combining the intriguing optical properties of gold nanoparticles with the inherent physical and dynamic properties of polymers can give rise to interesting hybrid nanomaterials. In this study, we report the synthesis of poly(acrylic acid) (PAA)-capped gold nanoparticles. The polyelectrolyte-wrapped gold nanoparticles were fully characterized and studied via a combination of techniques, i.e. UV-vis and infrared spectroscopy, dark field optical microscopy, SEM imaging, dynamic light scattering and zeta potential measurements. Although PAA-capped nanoparticles have been previously reported, this study revealed some interesting aspects of the colloidal stability and morphological change of the polymer coating on the nanoparticle surface in an electrolytic environment, at various pH values and at different temperatures.

A nanoparticle pseudo pathogen for rapid detection and diagnosis of virus infection.

We herein report a new rapid blood test for virus infection detection and diagnosis. A citrate gold nanoparticle is first coated with a virus lysate to form a gold nanoparticle pseudo pathogen. The gold nanoparticle pseudo virus is then mixed with a blood plasma or serum samples. If the blood sample is from a positive patient, the activated immune molecules in the blood such as antibodies, complement proteins and others will react with the nanoparticle pseudo virus, leading to nanoparticle aggregate formation. The nanoparticle aggregate formation is detected and measured using a particle sizing technique called dynamic light scattering. In this study, we applied this test for Zika virus infection detection. We tested blood plasma samples from 85 Zika positive patients, 40 Dengue positive patients, 10 Chikungunya positive patients, and 78 non-patient control samples collected from both endemic and non-endemic locations. The study shows that the new test has a higher sensitivity compared to some existing commercial tests in the market, while maintaining a similar specificity. Within 7 days from the symptom onset, the new test can detect 43% of the infected patients while a commercial anti-Zika IgM test detects only 26% of the infected patients. Within 14 days from the symptom onset, our new test detects 73% of the infected patients while the same commercial anti-Zika IgM test detects 53% of the infected patients. The test is extremely simple, easy to develop, with test results obtained within minutes. This new test platform may be potentially adapted for the detection and diagnosis of a wide range of viral infectious diseases, for example, the currently ongoing COVID-19.

A rapid blood test to monitor immunity shift during pregnancy and potential application for animal health management.

The immune health of a farm animal can have significant impact on its overall health, welfare and productivity. One of the most vulnerable physiological states for both humans and animals is pregnancy. Many systemic changes correlate with the gravid state, including shifts in the immune system that may impact the ability to respond optimally to pathogen challenge. Because of this, it would be beneficial to be able to monitor the immune health of the pregnant animals closely. Recently, we developed a new nanoparticle-enabled rapid blood test that can detect ongoing immune responses from both laboratory and farm animals. Here, we report that this novel test reveals highly repeatable and acute changes associated with pregnancy and peri-parturition period in laboratory mice and in cattle. We hypothesize that the test score change reflects changes in the immune status of the gravid females related to the humoral immune response. The test is easy to conduct, of low cost, with results obtained in less than 20 min. This rapid test could be potentially used as an onsite test in local farms and small clinics for animal health management.

Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies.

Dynamic light scattering (DLS) is an analytical tool used routinely for measuring the hydrodynamic size of nanoparticles and colloids in a liquid environment. Gold nanoparticles (GNPs) are extraordinary light scatterers at or near their surface plasmon resonance wavelength. In this study, we demonstrate that DLS can be used as a very convenient and powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies. The conjugation process between protein A and gold nanoparticles under different experimental conditions and the quality as well as the stability of the prepared conjugates were monitored and characterized systematically by DLS. Furthermore, the specific interactions between protein A-conjugated gold nanoparticles and a target protein, human IgG, can be detected and monitored in situ by measuring the average particle size change of the assay solution. For the first time, we demonstrate that DLS is able to directly and quantitatively measure the binding stoichiometry between a protein-conjugated GNP probe and a target analyte protein in solution.

Claudin-1 protein is a major factor involved in the tumorigenesis of colorectal cancer.

BACKGROUND: The molecular and morphological alterations of the tight junctions in colorectal cancer (CRC) are still poorly understood. The possible involvement of claudin-1 (CL-1), one of the major tight junctional proteins (TJPs), was investigated in the tumorigenesis of CRC. PATIENTS AND METHODS: Adenocarcinoma tissue and paired normal mucosa specimens were resected from surgical specimens of CRC patients and analyzed to determine whether the expression of CL-1 correlated with the clinicopathological factors and to determine the role of CL-1 in the alteration of tight junctions during tumorigenesis. RESULTS: The expression of CL-1 at the mRNA and protein levels was analyzed in 41 cases and was found to increase in the CRC tissue in comparison to that in the normal tissue specimens. The mRNA levels of CL-1 were correlated with tumor depth, but not with the preoperative carcinoembryonic antigen (CEA) serum level. When T84 cells, a human colon cancer cell line, were transfected with the CL-1 gene, the CL-1 overexpressing cells grew as aggregates in contrast to the monolayer formation of the parental cells. In addition, trypsin-treated CL-1 overexpressing cells aggregated more easily than did the parental cells. CONCLUSION: CL-1 plays a pivotal role in cell morphology and behavior in the colonic epithelium. CL-1 protein may therefore be one of the major factors involved in the tumorigenesis of CRC.

A Single-Step Gold Nanoparticle-Blood Serum Interaction Assay Reveals Humoral Immunity Development and Immune Status of Animals from Neonates to Adults.

A well-developed, functional immune system is paramount to combat harmful attacks from pathogenic organisms and prevent infectious diseases. Newborn animals and humans have only limited immunity upon birth, but their immune functions are expected to develop within weeks to months and eventually to reach a maturity that will provide full protection. Despite the importance of immune activity in animal and human health management, there is no convenient test available that allows for rapid assessment of the state of immune function in nonlaboratory settings. Here we report an extremely simple and rapid blood test that may be used in point-of-care clinics or field settings to evaluate the humoral immune status of animals. The test detects a cooperative interaction between a gold nanoparticle and arguably the three most important proteins involved in the immune system: immunoglobulin M (IgM), immunoglobulin G (IgG), and at least one complement protein, C3, in the blood serum. Such interactions cause the gold nanoparticles to form clusters and aggregates. The average particle size of the gold nanoparticle-serum mixture, measured by dynamic light scattering, corresponds positively to the immune status and activity of the subject. Our study demonstrates that the test may be used not only for monitoring the immune function development from neonates to adults, but also for detecting active immune responses during infection. Although data reported here are largely based on murine and bovine models, it is likely that this test will be applicable to humans as well.

A one-step highly sensitive method for DNA detection using dynamic light scattering.

A one-step homogeneous DNA detection method with high sensitivity was developed using gold nanoparticles (AuNPs) coupled with dynamic light scattering (DLS) measurement. Citrate-protected AuNPs with a diameter of 30 nm were first functionalized with two sets of single-stranded DNA probes and then used as optical probes for DNA detection. In the presence of target DNA, the hybridization between target DNA and the two nanoparticle probes caused the formation of nanoparticle dimers, trimers, and oligomers. As a result, the nanoparticle aggregation increased the average diameter of the whole nanoparticle population, which can be monitored simply by DLS measurement. A quantitative correlation can be established between the average diameter of the nanoparticles and the target DNA concentration. This DLS-based assay is extremely easy to conduct and requires no additional separation and amplification steps. The detection limit is around 1 pM, which is 4 orders of magnitude better than that of light-absorption-based methods. Single base pair mismatched DNAs can be readily discriminated from perfectly matched target DNAs using this assay.

A one-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering.

A one-step homogeneous immunoassay for the detection of a prostate cancer biomarker, free-PSA (prostate specific antigen), was developed using gold nanoparticle probes coupled with dynamic light scattering (DLS) measurements. A spherical gold nanoparticle with a core diameter around 37 nm and a gold nanorod with a dimension of 40 by 10 nm were first conjugated with two different primary anti-PSA antibodies and then used as optical probes for the immunoassay. In the presence of antigen f-PSA in solution, the nanoparticles and nanorods aggregate together into pairs and oligomers through the formation of a sandwich type antibody-antigen-antibody linkage. The relative ratio of nanoparticle-nanorod pairs and oligomers versus individual nanoparticles was quantitatively monitored by DLS measurement. A correlation can be established between this relative ratio and the amount of antigen in solution. The light scattering intensity of nanoparticles and nanoparticle oligomers is several orders of magnitude higher than proteins and other typical molecules, making it possible to detect nanoparticle probes in the low picomolar concentration range. f-PSA in the concentration range from 0.1 to 10 ng/mL was detected by this one-step and washing-free homogeneous immunoassay.

Surface modification of gold nanorods through a place exchange reaction inside an ionic exchange resin.

A place exchange reaction between 11-mercaptoundecanoic acids (MUA) and cetyltrimethylammonium bromide (CTAB)-protected gold nanorods (GNRs) was conducted inside an ionic exchange resin; this modification resulted in functional gold nanorods soluble in both polar and nonpolar organic solvents.

Recombinant human monoclonal igA antibody against CEA to recruit neutrophils to CEA-expressing cells.

IgA is able to trigger antibody-dependent cellular cytotoxicity (ADCC) by recruiting neutrophils expressing the Fc receptor for the CHalpha chain. We herein describe the preparation of a human recombinant anti-CEA IgA antibody to kill carcinoembryonic antigen (CEA)-expressing tumor cells via ADCC by neutrophils. A single chain Fv (scFv) gene was constructed using a cDNA library of a hybridoma clone that produces a human anti-CEA monoclonal IgG4 (C2-45). The scFv gene, linked with a CHalpha gene, was inserted into the pBK283 vector, which was cotransfected into BmN4 insect cells with the wild-virus BmNPV. After cloning and amplification, the recombinated virus was injected into silkworm larvae. The resulting human recombinant IgA, designated as 45scFvLCHalpha, was purified from hemolymph by Ni-affinity chromatography and characterized by ELISA, Western blotting, and the ADCC assay. 45scFvLCHalpha with an IgA antigenicity was bound to CEA and showed effective killing of the CEA-expressing cells in the presence of IFN-gamma-activated neutrophils. These data suggest the recombinant anti-CEA IgA antibody recruiting neutrophils maybe a useful means for the antibody-based immunotherapy of human CEA-expressing tumors.

Photothermal ablation of amyloid aggregates by gold nanoparticles.

Amyloid peptide (Abeta) is found in the brain and blood of both healthy and diseased individuals alike. However, upon secondary structure transformation to a beta-sheet dominated conformation, the protein aggregates. These aggregates accumulate to form neuritic plaques that are implicated in the pathogenesis of Alzheimer's disease. Gold nanoparticles are excellent photon-thermal energy converters. The extinction coefficient of the surface plasmon band of gold nanoparticles is very large when compared to typical organic dyes. In this study, gold nanoparticle-Abeta conjugates were prepared and the photothermal ablation of amyloid peptide aggregates by laser irradiation was studied. Monofunctional gold nanoparticles were prepared using a recently reported solid phase modification method and then coupled to fragments of Abeta peptide, namely Abeta(31-35) and Abeta(25-35). The conjugates were then mixed with Abeta fragments in solution. The aggregated peptide formation was studied by a series of spectroscopic and microscopic techniques. The peptide aggregates were then irradiated by a continuous laser. With gold nanoparticle-Abeta conjugates present the aggregates were destroyed by photothermal ablation. Gold nanoparticles without Abeta conjugation were not incorporated into the aggregates and when irradiated did not result in photothermal ablation. With gold nanoparticle-Abeta conjugates the ablation was selective to the site of irradiation and minimal damage was observed as a result of thermal diffusion. In addition to the application of photoablation to a protein-based sample the nanoparticles and the chemistry involved provide an easily monofunctionalized photothermal material for the biological conjugation.