One-Component Dual-Readout Aggregation-Induced Emission Nanobeads for Qualitative and Quantitative Detection of C-Reactive Protein at the Point of Care.

Fluorescent lateral flow immunoassay (LFA) systems are versatile tools for sensitive and quantitative detection of disease markers at the point of care. However, traditional fluorescent nanoparticle-based lateral flow immunoassays are not visible under room light, necessitate an additional fluorescent reader, and lack flexibility for different application scenarios. Herein, we report a dual-readout LFA system for the rapid and sensitive detection of C-reactive protein (CRP) in clinical samples. The system relied on the aggregation-induced emission nanobeads (AIENBs) encapsulated with red AIE luminogen, which possesses both highly fluorescent and colorimetric properties. The AIENB-based LFA in the naked-eye mode was able to qualitatively detect CRP levels as low as 8.0 mg/L, while in the fluorescent mode, it was able to quantitatively measure high-sensitivity CRP (hs-CRP) with a limit of detection of 0.16 mg/L. The AIENB-based LFA system also showed a good correlation with the clinically used immunoturbidimetric method for CRP and hs-CRP detection in human plasma. This dual-modal AIENB-based LFA system offers the convenience of colorimetric testing and highly sensitive and quantitative detection of disease biomarkers and medical diagnostics in various scenarios.

Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

Immunoengineered magnetic-quantum dot nanobead system for the isolation and detection of circulating tumor cells.

BACKGROUND: Highly efficient capture and detection of circulating tumor cells (CTCs) remain elusive mainly because of their extremely low concentration in patients' peripheral blood. METHODS: We present an approach for the simultaneous capturing, isolation, and detection of CTCs using an immuno-fluorescent magnetic nanobead system (iFMNS) coated with a monoclonal anti-EpCAM antibody. RESULTS: The developed antibody nanobead system allows magnetic isolation and fluorescent-based quantification of CTCs. The expression of EpCAM on the surface of captured CTCs could be directly visualized without additional immune-fluorescent labeling. Our approach is shown to result in a 70-95% capture efficiency of CTCs, and 95% of the captured cells remain viable. Using our approach, the isolated cells could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. We applied iFMNS for testing CTCs in peripheral blood samples from a lung cancer patient. CONCLUSIONS: It is suggested that our iFMNS approach would be a promising tool for CTCs enrichment and detection in one step.

Silver-amplified fluorescence immunoassay via aggregation-induced emission for detection of disease biomarker.

Development of simple, robust, and reliable detection strategy of disease biomarkers holds tremendous promise for early clinical diagnosis and prognosis of diseases. In this work, through combining a silver nanoparticle (AgNP) linked immunoassay and aggregation induced emission (AIE)-based fluorogenic Ag+ probe, we developed a silver-amplified fluorescence immunoassay for the detection of disease biomarkers. This method overcame the intrinsic limitations of enzymes as the dissolution of AgNPs generated numerous Ag+, which could switch on the fluorogenic Ag+ probe driven by tetrazolate-Ag+ complexation. As a proof of concept, our method could be used for determining α-fetoprotein (AFP) with a linear relationship in concentrations ranging from 0.1 ng mL-1 to 5 µg mL-1 and a low limit of detection of 42 pg mL-1. Our method was successfully confirmed for the detection of AFP in real serum samples from hepatocellular carcinoma (HCC) patients, demonstrating the great potential for clinical diagnosis.

Bimodal size distribution immuno-quantum dots for fluorescent western blotting assay with high sensitivity and extended dynamic range.

A highly sensitive quantum dot (QD)-based western blot assay with extended dynamic range was developed. Bimodal size distribution QD (BQ) immunoprobes composed of small size single QD (7.3 nm) and big size QD nanobead (QB) (82.9 nm) were employed for fluorescent western blot immunoassay on a membrane. Small size QD immunoprobes contributed to wider dynamic range of assay, while big size QB immunoprobes provided higher detection sensitivity. This BQ-based western blot assay can achieve a wide dynamic range (from 7.8 to 4000 ng IgG) and is nearly as sensitive as commercial available ultrasensitive chemiluminescent methods, just using a simple gel imager with UV light (365 nm) excitation and red light filter (610 nm). The fluorescent signals of BQ western blot were stable for 10 min, while chemiluminescent signals faded after 1 min. Moreover, this BQ immunoprobe was utilized for the detection of housekeeping protein and specific target proteins in complex cell lysate samples. The limit of detection of housekeeping protein is 0.25 µg of cell lysate, and the signal intensities were proportional to loading protein amount in a wide range from 0.61 to 80 µg. We believe that this new strategy of bimodal size distribution nanoparticles can also be expanded for other functional nanoparticle-based biological assays to improve the sensitivity and extend the dynamic range. Graphical abstract.

Plasmon-Induced Photoreduction System Allows Ultrasensitive Detection of Disease Biomarkers by Silver-Mediated Immunoassay.

Current strategies for the detection of disease biomarkers often require enzymatic assays that may have limited sensitivity due to inferior stability and vulnerable catalytic activity of the enzyme. A new enzyme-free amplification method for identifying suitable biomarkers is necessary to lower the limit of detection and improve many critical diagnosis applications. Here, we presented an enzyme-free amplified plasmonic immunoassay that enhanced the detection sensitivity of disease biomarkers by combining a novel plasmon-induced silver photoreduction system with a silver nanoparticle (AgNP)-linked immunoassay. The key step to achieving ultrasensitivity was to use Ag+ from dissolved AgNPs that control the growth rate of the silver coating on plasmonic nanosensors under visible light illumination. We demonstrated the outstanding sensitivity and robustness of this assay by detecting the disease biomarker alpha-fetoprotein (AFP) at a low concentration of 3.3 fg mL-1. The detection of AFP was further confirmed in the sera of hepatocellular carcinoma patients.

Enzyme-free amplified SERS immunoassay for the ultrasensitive detection of disease biomarkers.

We developed a novel enzyme-free amplified SERS immunoassay by combining silver nanoparticle (AgNP)-linked immunoreaction and SERS transduction for the detection of disease biomarkers. As a proof of concept, our method was successfully illustrated with the disease biomarker α-fetoprotein with the detection limit of 3.3 × 10-13 g mL-1 and a double-blind experiment consisting of tens of serum samples was performed to confirm its reliability.

A Hallmark-Based Six-Gene Expression Signature to Assess Colorectal Cancer and Its Recurrence Risk.

Purpose: As part of the effort to establish a general profile for solid tumors, the aim of this study was to develop a real-time polymerase chain reaction (RT-PCR)-based assay to assess colorectal cancer (CRC) and its recurrence risk utilizing the limited amounts of tissues available from biopsies through colonoscopy. Materials and Methods: Six candidate genes, reflecting the hallmarks of cancer cells, were identified by analyzing the gene expression profiles of primary invasive tumors in the public database. The expression of these genes in CRC and noncancerous colon tissues was quantified by RT-quantitative PCR. Classifiers were then generated to distinguish the tumors from the normal colon tissues, and to assess the risk of CRC recurrence based on the disease-free survival time, overall survival time, and metastatic status of the patients. Results: The expression profile of a five-gene panel was utilized to build a model that is capable of distinguishing CRC cancer tissues from noncancerous colorectal tissues (p < 0.0001). A classifier based on the expression signature of four genes, three of which were included in the five-gene panel, was then developed for assessing the tumor recurrence risk. This classifier could correctly identify those with a poor likelihood of survival (high risk of recurrence) >80% of time. There was a significant difference in disease-free survival time between patients in the low recurrence group and those in the high-risk group. Conclusion: The expression signatures of the six genes that reflect the genetic hallmarks of cancer cells could serve as a biomarker for identifying CRC and assessing the risk of recurrence with high sensitivity and specificity.

Blended gold/MnO2@BSA nanoparticles for fluorometric and magnetic resonance determination of ascorbic acid.

A fluorometric and magnetic resonance (MR) dual-modal detection scheme is presented for determination of ascorbic acid (AA). It is based on the use of a blended Au/MnO2@BSA mixture that was prepared via a biomimetic strategy, using bovine serum albumin (BSA) as the template at physiological temperature. The MnO2@BSA fraction (one part of the composite) is not susceptible to MR but can be degraded to MR-active compounds upon a redox reaction with even ultralow concentrations of AA. In parallel, the blended Au/MnO2@BSA recovers its fluorescence because MnO2@BSA acts as a quencher of the fluorescence of circumjacent Au@BSA (the other part of the composite). Fluorescence typically is measured at excitation/emission wavelengths of 470/625 nm. Leveraging on this redox reaction between MnO2 and AA, a dual-mode detection scheme for AA was developed. Both the fluorescence and the MR signal increase with the concentration of AA. The lowest limit for the detection of AA is 0.6 µM in the fluorometric mode and 0.4 µM in the MR mode. Analysis of AA-spiked serum samples showed that the recoveries obtained by either the fluorometric and MR mode can reach 94%. This is the first report of the use of blended nanoparticles with their inherent cross-validation regularity. Graphical abstract Schematic presentation of the biomimetic synthesis of blended Au/MnO2@BSA nanoprobes and fluorometric/MR cross-validation dual-modal detection of ascorbic acid.

Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers.

The incidence of aggressive neuroendocrine prostate cancers (NEPC) related to androgen-deprivation therapy (ADT) is rising. NEPC is still poorly understood, such as its neuroendocrine differentiation (NED) and angiogenic phenotypes. Here we reveal that NED and angiogenesis are molecularly connected through EZH2 (enhancer of zeste homolog 2). NED and angiogenesis are both regulated by ADT-activated CREB (cAMP response element-binding protein) that in turn enhances EZH2 activity. We also uncover anti-angiogenic factor TSP1 (thrombospondin-1, THBS1) as a direct target of EZH2 epigenetic repression. TSP1 is downregulated in advanced prostate cancer patient samples and negatively correlates with NE markers and EZH2. Furthermore, castration activates the CREB/EZH2 axis, concordantly affecting TSP1, angiogenesis and NE phenotypes in tumor xenografts. Notably, repressing CREB inhibits the CREB/EZH2 axis, tumor growth, NED, and angiogenesis in vivo. Taken together, we elucidate a new critical pathway, consisting of CREB/EZH2/TSP1, underlying ADT-enhanced NED and angiogenesis during prostate cancer progression.

Sensitive detection of protein biomarkers using silver nanoparticles enhanced immunofluorescence assay.

Detection of biomarkers is extremely important for the early diagnosis of diseases. Here, we developed an easy and highly sensitive fluorescence detection system for the determination of biomarkers by combining the rapid separation of magnetic beads and silver nanoparticles labeled antibodies. An ultrasensitive silver ions fluorescence probe 3', 6'-bis (diethylamino)-2-(2-iodoethyl) spiro[isoindoline-1, 9'-xanthen]-3-one (Ag+-FP) was applied to immunoassay. A significant signal amplification was achieved as the AgNPs can be dissolved by H2O2 and generate numerous Ag+, which would turn "on" the fluorescence of Ag+-FP. Using α-fetoprotein (AFP) and C-reactive protein (CRP) as target analytes, good linear responses were obtained from 0.1 to 10 ng mL-1 and the limits of detection (LOD) were as low as 70 pg·mL-1 and 30 pg·mL-1, respectively. In addition, the developed system was further evaluated for the detection of real samples including 30 positive serum specimens obtained from hepatocarcinoma patients and 20 negative serum samples, and performs as well as the commercial electrochemiluminescence immunoassay (ECLI) method with less cost and more convenience. Thus, the designed detection system can be used as a promising platform for the detection of a variety of biomarkers and served as a powerful tool in clinical diagnosis.

Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum.

Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL(-1), 0.1 ng mL(-1) and 1 ng mL(-1) respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively.

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay.

Convenient and rapid immunofiltration assays (IFAs) enable on-site "yes" or "no" determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.

AURKA upregulation plays a role in fibroblast-reduced gefitinib sensitivity in the NSCLC cell line HCC827.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat non-small cell lung carcinoma (NSCLC) patients that have EGFR-activating mutations. EGFR-TKI monotherapy in most NSCLC patients with EGFR mutations who initially respond to EGFR-TKIs results in the development of acquired resistance. We investigated the role of fibroblasts in stromal cell-mediated resistance to gefitinib-induced apoptosis in EGFR-mutant NSCLC cells. While gefitinib induced apoptosis in EGFR-mutant NSCLC cells, apoptosis induction was diminished under stromal co-culture conditions. Protection appeared to be mediated in part by Aurora-A kinase (AURKA) upregulation. The protective effect of stromal cells was significantly reduced by pre-exposure to AURKA-shRNA. We suggest that combinations of AURKA antagonists and EGFR inhibitors may be effective in clinical trials targeting mutant EGFR NSCLCs.

Simple and sensitive detection of HBsAg by using a quantum dots nanobeads based dot-blot immunoassay.

Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection method for the detection of infectious disease markers that couples the dot-blot immunoassay with quantum dots nanobeads (QDNBs) as a reporter. First, the QDNBs were prepared by an oil-in-water emulsion-evaporation technique. Because of the encapsulation of several QDs in one particle, the fluorescent signal of reporter can be amplified with QDNBs in a one-step test and be read using a UV lamp obviating the need for complicated instruments. Detection of disease-associated markers in complex mixture is possible, which demonstrates the potential of developing QDNBs into a sensitive diagnostic kit.

Identification of IFN-γ-producing innate B cells.

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a(hi)FcγRIII(hi) B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton's tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.

The protective effect of PNU-282987, a selective α7 nicotinic acetylcholine receptor agonist, on the hepatic ischemia-reperfusion injury is associated with the inhibition of high-mobility group box 1 protein expression and nuclear factor κB activation in mice.

Hepatic ischemia-reperfusion (I/R) injury contributes to hepatic dysfunction and failure after liver transplantation, major hepatic resection, trauma, and hypovolemic shock. Therefore, reducing I/R injury is an important goal to improve the outcome of these procedures. Recently, high-mobility group box 1 protein (HMGB1) has been identified as a pathogenic mediator in several inflammatory diseases, including hepatic I/R. PNU-282987, a selective α7 nicotinic acetylcholine receptor agonist, prevents nuclear factor κB (NF-κB) activation and thereby inhibits cytokine secretion through a specific cholinergic anti-inflammatory pathway. Our study was designed to evaluate whether PNU-282987 would inhibit HMGB1 expression and prevent I/R-induced liver damage. C57BL/6 mice were randomly divided into 3 groups as follows: sham group, vehicle plus I/R group, and PNU-282987 plus I/R group. Mice were subjected to 70% partial hepatic I/R for 60 min and pretreated with either vehicle or with PNU-282987, and blood and hepatic tissue samples were collected at 3, 6, and 12 h following reperfusion. The results showed that pretreatment with PNU-282987 decreased serum transaminase levels and ameliorated liver injury after hepatic I/R. Moreover, pretreatment with PNU-282987 suppressed NF-κB activation, cytokine production (tumor necrosis factor α, interleukin 1ß), and HMGB1 expression in liver after hepatic I/R. These observations suggest that PNU-282987 protects the liver from I/R injury possibly by inhibiting HMGB1 expression, suppressing cytokine production, and preventing NF-κB activation in mice.

Tyrosine kinase Btk is required for NK cell activation.

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk(-/-) NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk(-/-) mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk(-/-) mice after the adoptive transfer of Btk(+/+) NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response.

Combination of Poly I:C and arsenic trioxide triggers apoptosis synergistically via activation of TLR3 and mitochondrial pathways in hepatocellular carcinoma cells.

Poly I:C (polyinosinic acid:polycytidylic acid), an analogue of dsRNA (double-stranded RNA), can lead to apoptosis in human cancer cells and has been used as an adjuvant to treat cancer patients. ATO (arsenic trioxide) is used effectively in the treatment of HCC (hepatocellular carcinoma). We sought to evaluate whether Poly I:C could enhance the potentiation of ATO in HCC. Combination of Poly I:C and ATO synergistically inhibited the growth of SMMC-7721 cells. Treatment with Poly I:C alone or combined with ATO-activated TLR3 (Toll-like receptor 3) pathway, increased ROS (reactive oxygen species) generation and mitochondrial dysfunction. The combined treatment also caused caspase-3, -8, -9 activation. Moreover, the combined therapy caused Bcl-2 and survivin down-regulation, Bax up-regulation and Bid activation. In conclusion, the Poly I:C and ATO combination is potentially a novel and effective approach for the treatment of HCC.