Colorimetric Enzyme-linked Immunosorbent Assay Research Articles

Biorecognition and detection of antigens from Mycobacterium tuberculosis using a sandwich ELISA associated with magnetic nanoparticles

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the 10 leading causes of death worldwide, especially in low-income areas. A rapid, low-cost diagnostic assay for TB with high sensitivity and specificity is not currently available. Bio-functionalized magnetic nanoparticles (MNPs) which are able to efficiently detect and concentrate biomolecules from complex biological samples, allows improving the diagnostic immunoassays. In this way, a proof-of-concept of MNP-based sandwich immunoassay was developed to detect various MTB protein antigens. The superficial and secretory antigenic proteins considered in this research were: CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B, and MoeX. The proteins were cloned and expressed in an E. coli system. Polyclonal antibodies (ab) against the recombinant antigens were elicited in rabbits and mice. Antibodies were immobilized on the surface of amine-silanized nanoparticles (MNP@Si). The functionalized MNP@Si@ab were tested in a colorimetric sandwich enzyme-linked immunosorbent assay (sELISA-MNP@Si@ab) to recognize the selected antigens in sputum samples. The selected MTB antigens were successfully detected in sputum from TB patients in a shorter time (~ 4 h) using the sELISA-MNP@Si@ab, compared to the conventional sELISA (~15 h) standardized in home. Moreover, the sELISA-MNP@Si@ab showed the higher sensitivity in the real biological samples from infected patients.

Immunological aspects of bacterial keratites in patients with diabetes mellitus

Background. The purpose was to analyze the cytokine balance of lacrimal fluid in patients with bacterial keratitis and diabetes mellitus (DM) at the first visit and to identify the immunological aspects of the disease. Materials and methods. The analysis of pro- and anti-inflammatory cytokine concentration in the lacrimal fluid was performed in 17 patients with type 1 DM and bacterial keratitis and 15 nondiabetic patients with bacterial keratitis at the first visit. Data from 14 healthy individuals were used for comparison. In addition to standard ones, ophthalmic examination methods included bacteriological examination, fluorescein test, anterior segment optical coherence tomography, non-contact corneal aesthesiometry. The levels of interleukin (IL) 1β, IL-6 and IL-10 in the lacrimal fluid of the sick and the contralateral eye were determined by a quantitative colorimetric enzyme-linked immunosorbent assay using ELISA kits. Results. In DM patients with bacterial keratitis, the concentration of IL-1β and IL-6 in the lacrimal fluid of the sick eye exceeded that in healthy individuals (p < 0.05) and did not differ significantly from nondiabetic patients with bacterial keratitis (p > 0.05). In the lacrimal fluid of the contralateral eye of DM patients with bacterial keratitis, the level of IL-1β and IL-6 exceeded the corresponding indicators of nondiabetic patients with bacterial keratitis and healthy individuals (p < 0.05). The concentration of IL-10 in the lacrimal fluid of the contralateral eye in DM patients with bacterial keratitis exceeded that in healthy individuals (p < 0.05) and did not significantly differ from those in nondiabetic patients with bacterial keratitis (p > 0.05). Conclusions. DM patients with bacterial keratitis have immunological features of the disease.

A Portable, Cost-Effective and User-Friendly Instrument for Colorimetric Enzyme-Linked Immunosorbent Assay and Rapid Detection of Aflatoxin B1.

Food analysis based on the enzyme-linked immunosorbent assay (ELISA) is simple, sensitive and rapid, but requires a costly colorimetric instrument. The aim of this work was to develop a portable, low-cost and user-friendly colorimetric instrument for colorimetric ELISA and aflatoxin B1 (AFB1) detection. The principle of the developed instrument was employing a light-emitting diode to generate the signal light and using a light-dependent resistor to measure the signal light absorbed by the oxidized 3,3′,5,5′-tetramethyl benzidine. The absorption spectra revealed that the solution absorbed signal light more strongly after reaction with H2SO4, and blue light would be favorably absorbed. Evaluations on the stability and accuracy of the instrument and interference from ambient light showed that the fabricated instrument was stable, accurate, capable of quantitative detection and insensitive to ambient light changes. In addition, this instrument is user-friendly since it could calculate and report the final amount of AFB1 to the operator. Measurements of maize and peanuts showed that the instrument provided as accurate results as the professional equipment. With the low fabrication cost (about RMB 129 or USD 20), portability, and user-friendliness, this instrument presents attractive potential in the rapid detection of AFB1.

PECULIARITIES OF THE LACRIMAL FLUID CYTOKIN PROFILE IN PATIENTS WITH BACTERIAL KERATITITIS OF THE II DEGREE SEVERETY AND DIABETES MELLITUS

Summary. The aim was to identify the features of the lacrimal fluid cytokine profile in patients with bacterial keratitis of the II degree of severity and type 1 DM in the dynamics of observation.
 Materials and methods. The lacrimal fluid cytokine profile was analyzed in 6 patients with type 1 DM and bacterial keratitis of the II degree of severity and 6 nondiabetic patients with bacterial keratitis of the II degree of severity on the 1st day, 10-14th day and 24th day of observation. Data from 14 healthy individuals of the appropriate age were used for comparison. II degree of severity of bacterial keratitis was determined according to our proposed scheme at a sum of points 15-21. All patients were treated with instillations of ofloxacin, antiseptics, antioxidants, reparents, artificial tears, mydriatics, systemic anti-inflammatory therapy. The concentration of interleukins (IL)-1β, IL-6 and IL-10 in the lacrimal fluid of the sick and the contralateral eye was determined by quantitative colorimetric enzyme-linked immunosorbent assay using ELISA kits.
 Results and discussion. In patients with bacterial keratitis of the II degree of severity, both with DM and nondiabetic, within 10-14 days of observation invented the expression of proinflammatory cytokines IL-1β and IL-6 in the lacrimal fluid of the sick eye and anti-inflammatory cytokine IL-10 - in lacrimal fluid of the contralateral eye (p<0.05). In DM patients, a longer expression of IL-1β and IL-6 was detected in the lacrimal fluid of the sick and the contralateral eye, which was observed on the 24th day of the study (p<0.05).
 Conclusions. There are features of the lacrimal fluid cytokine profile in DM patients with bacterial keratitis of the II severity in the dynamics of observation, namely the longer expression of proinflammatory cytokines IL-1β, IL-6 in the sick and the contralateral eye.

Smartphone-based fluorescent ELISA with simple fluorescent enhancement strategy for Opisthorchis viverrini (Ov) antigen detection in urine samples

Enzyme-linked immunosorbent assay (ELISA) is a new approach for the diagnosis of Opisthorchis viverrini antigen (OvAg) in urine samples. Herein, we demonstrate a portable fluorometer based on the smartphone to use as the point-of-care device for the diagnosis of OvAg in urine samples by fluorescence ELISA. In addition, the detection sensitivity of the fluorescent assay has been improved by a simple strategy using aqueous micelles of the non-ionic surfactant. The combination of a smartphone-based device with fluorescent enhancement provides an excellence tool for the diagnosis of the OvAg in urine samples. The smartphone-based device shows excellent potential for measuring the fluorescence signal comparable with a fluorescence microplate reader. The signal in the green channel (G) of the smartphone representing the emission intensity from the fluorescence ELISA exhibits a good linear relationship with OvAg concentration with an LOD of 33 ng mL−1. The proposed device achieved high diagnostic sensitivity, specificity, and accuracy of 84.88%, 89.66%, and 86.14%, respectively, compared to the measurement by conventional instruments (colorimetric ELISA). The smartphone-based device has been verified for the diagnosis of OvAg in urine samples from an endemic area with excellent results. Therefore, smartphone-based diagnostics is an effective point-of-care device for on-site assay of OvAg detection in urine samples.

Аналіз цитокінового балансу в сльозовій рідині у хворих на бактеріальний кератит і цукровий діабет залежно від тяжкості захворювання

The aim. To analyze the cytokine balance of tear fluid in patients with bacterial keratitis at presentation depending on the severity of the disease and the presence of diabetes mellitus (DM). Materials and methods. The analysis was performed through the comparison of the level of pro- and anti-inflammatory cytokines in the tear fluid of 17 patients with type 1 DM and bacterial keratitis and 15 patients with bacterial keratitis without DM at presentation. Data from 14 healthy individuals of the appropriate age were also used for comparison. The patients with bacterial keratitis were divided into subgroups according to the severity of bacterial keratitis. The levels of IL-1β, IL-6 and IL-10 in the tear fluid of the sick and the contralateral eye were determined by quantitative colorimetric enzyme-linked immunosorbent assay. Results and discussion. At presentation, patients with bacterial keratitis, both with and without DM, showed increased levels of proinflammatory cytokines, namely IL-1β and IL-6, in the tear fluid of the sick eye, which correlated with the severity of the disease, and also increased level of anti-inflammatory cytokine IL-10 in the tear fluid of the contralateral eye. In addition, the concentrations of proinflammatory cytokines IL-1β, IL-6 in the tear fluid of the contralateral eye in DM patients were increased at all degrees of severity of bacterial keratitis. Conclusions. In patients with bacterial keratitis, the cytokine balance of the tear fluid of the sick and the contralateral eye depends on the severity of the disease and the presence of DM. Keywords: diabetes mellitus, bacterial keratitis, severity of keratitis, cytokines, interleukins.

IMMUNOLOGICAL FEATURES OF THE DYNAMICS OF THE I DEGREE SEVERETY BACTERIAL KERATITES DEPENDING ON THE PRESENCE OF DIABETES MELLITUS

Summary. In patients with diabetes mellitus (DM) there are features of the dynamics of the I degree severity bacterial keratitis, leading to prolongation of the disease and worsening of its consequences.
 The aim was to identify immunological features of the dynamics of the I degree severity bacterial keratitis depending on the presence of type 1 DM.
 Materials and methods. The level of interleukins (IL) was analyzed, namely IL-1β, IL-6 and IL-10 in the lacrimal fluid of the sick and the contralateral eye of 6 patients with type 1 DM and the I degree severity bacterial keratitis and 6 nondiabetic patients with I degree severity bacterial keratitis on the 1st day, 10-14th day and 24th day of observation. Data from 14 healthy individuals of the appropriate age were used for comparison. The severity of bacterial keratitis was determined according to our scheme at a total score of ≤ 14. All patients were treated with instillations of ofloxacin, antiseptics, antioxidants, reparents, artificial tears, mydriatics, systemic anti-inflammatory therapy. The level of IL in the lacrimal fluid was determined by quantitative colorimetric enzyme-linked immunosorbent assay using ELISA kits by ANOGEN according to the instructions for use.
 Results and discussion. In patients with I degree severity bacterial keratitis, both DM and nondiabetic, on the 1st day of observation there was an increase in the level of IL-1β and IL-6 in the lacrimal fluid of the sick eye and IL-10 – of the contralateral eye compared to healthy persons, on the 10th-14th day — increase in the level of IL-1β and IL-6 of the sick and the contralateral eye, as well as IL-10 – of the contralateral eye (p<0.05). On the 24th day in nondiabetic patients with I degree severity bacterial keratitis normalization of interleukin levels in the lacrimal fluid of the sick and the contralateral eye was found, but in DM patients remained elevated levels of IL-1β, IL-6, both in the sick and in the contralateral eye (p<0.05).
 Conclusions. In DM patients there are immunological features of the dynamics of the I degree severity bacterial keratitis, namely a longer increase in the level of proinflammatory cytokines IL-1β, IL-6 in the lacrimal fluid of the sick and the contralateral eye.

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins

Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.

Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.

Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies. The ELISA data confirmed the nanobody specificity but showed that the saturation of the fluorescence signal already in the presence of few hundreds of cells limited the dynamic range of the method. As an alternative, Avi-tagged nanobodies were used in combination with streptavidin-linked horseradish peroxidase for developing a diagnostic colorimetric cell ELISA, the limit-of-detection of which was 3.2 and 4.5 cells/ml for the two tested cyanobacteria strains, whereas the linear range of the assay was expanded from 10 to 10,000 cells. The fluorescent nanobodies were finally exploited for quantifying cyanobacteria by thermal lens spectrometry (TLS) that enabled to reach a limit-of-detection of 1.2 cells/ml and provided a linear range of measurement between 0 and 10,000 cells. No cross-reactivity with unrelated microalgae was detected and both colorimetric ELISA and TLS provided a linear range of detection of few logs. The data indicate that nanobodies are suitable capture reagents and that both TLS and colorimetric ELISA are reliable to monitor variations of cyanobacteria populations.

Nanobody multimerization strategy to enhance the sensitivity of competitive ELISA for detection of ochratoxin A in coffee samples

Enzyme-linked immunosorbent assay (ELISA) is an ideal immunoassay technique for large scale screening detection of various chemical contaminants in food due to its high throughput, low cost and ease of automation. However, the widespread use of traditional colorimetric ELISA has been hindered by the relatively low sensitivity. In this paper, the OTA-specific nanobody (Nb28) was fused with the C4-binding protein (C4bp) C-terminal fragment to form a self-assembled heptamer fusion protein (Nb28-C4bpα). In addition, the heptamer with higher affinity was employed to develop an ultrasensitive competitive ELISA for the detection of OTA in coffee samples. The effect of this sensitivity enhancement strategy was demonstrated with the IC50 of 0.13 ng/mL and LOD of 0.009 ng/mL for the heptamer-based ELISA, which is 26.54-fold and 175.56-fold lower than that of the monomer-based ELISA, respectively. The proposed nanobody multimerization can be a particularly appealing strategy to enhance the sensitivity or analytical signals in various immunoassays.

NanoLuc luciferase as a suitable fusion partner of recombinant antibody fragments for developing sensitive luminescent immunoassays

Enzyme-linked immunosorbent assays (ELISAs) are essential for monitoring various biomarkers. Competitive and noncompetitive (sandwich) assay formats are used to determine hapten and macromolecule levels, respectively. Both formats require more sensitive detection of reporter enzymes for greater assay sensitivities. We previously reported the utility of wild-type Gaussia luciferase (wtGLuc) as a fusion partner with antibody single-chain Fv fragments (scFvs) for developing sensitive luminescent ELISAs. Here, we evaluated utility of NanoLuc luciferase (NLuc), a recently developed luciferase, as fusion partner with scFvs from the view of comparison with wtGLuc and a mutant of alkaline phosphatase (ALP’). Thyroxine (T4) and T4-labeled albumin were chosen as model haptenic and macromolecular antigens, respectively. An in-house-prepared anti-T4 scFv was fused with NLuc, wtGLuc, or ALP’. The scFv−NLuc fusion protein showed 47-fold and 29-fold lower limit of detection [LOD; 59 zmol (per assay)] than the wtGLuc- and ALP’-fusions, respectively. In a competitive T4 ELISA, the NLuc-fusion showed 9.3- and 6.3-fold lower LOD, (0.67 pg) than the wtGLuc- and ALP’-fusions, respectively, with a higher specificity in clinical applications. A typical colorimetric ELISA using a peroxidase-labeled second antibody showed 70-fold higher LOD than NLuc-based ELISA. Another advantage of the NLuc-fusion was shown in the sandwich assays; the LOD of T4-labeled albumin (5.0 fmol) was >6-fold lower than that of the other luminescent ELISAs. In an additional sandwich assay developed to count bacteriophage particles, NLuc enabled more sensitive determination than wtGLuc, whereas ALP’ showed nearly equivalent performance. Its slowest alteration rate for light intensity after starting the enzyme reaction should enable robust batch-by-batch assay operations. Thus, we concluded that scFv−NLuc fusions serve as suitable probes in various types of immunoassays and may facilitate higher sensitivities with practical specificities.

An electricity- and instrument-free infectious disease sensor based on a 3D origami paper-based analytical device.

Infectious diseases cause millions of deaths annually in the developing world. Recently, microfluidic paper-based analytical devices (μPADs) have been developed to diagnose such diseases, as these tests are low cost, biocompatible, and simple to fabricate. However, current μPADs are difficult to use in resource-limited areas due to their reliance on external instrumentation to measure and analyze the test results. In this work, we propose an electricity and external instrumentation-free μPAD sensor based on the colorimetric enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infectious disease (3D-tPADs). Designed based on the principle of origami, the proposed μPAD enables the sequential steps of the colorimetric ELISA test to be completed in just ∼10 min. In addition, in order to obtain an accurate ELISA result without using any instrument, we have integrated an electricity-free "timer" within the μPAD that can be controlled by the buffer viscosity and fluid path volume to indicate the appropriate times for washing and color development steps, which can avoid false positive or false negative results caused by an extended or shortened amount of washing and development times. Due to the low background noise and high positive signal intensity of the μPAD, positive and negative detection results can be distinguished by just the naked eye. Furthermore, the ELISA result can be semi-quantified by comparing the results shown on the μPAD with a color chart diagram with a detection limit of HIV type 1(HIV-1) p24 antigen as low as 0.03 ng mL-1. These results demonstrate the proposed sensor can perform infectious disease diagnosis without external instrumentation or electricity, extending the application of the μPAD test for on-site detection and use in resource-limited settings.

PH Responded Colorimetric ELISA for Sensitive Detection of Salmonella Choleraesuis in Milk

Human maybe get poisoning after eating Salmonella choleraesuis-infected food. Thus,developing a new analytical technology for rapid and sensitive detection of Salmonella choleraesuis in food is urgently needed. Hence,a pH responded colorimetric enzyme-linked immunosorbent assay(pH-ELISA)was developed for sensitive detection of Salmonella choleraesuis in milk. In this method,glucose oxidase(GOx)was used to catalyze glucose for the generation of gluconic acid. The color of bromocresol purple(BCP)was changed from purple to bright yellow because the pH of sample solution decreased. Under the optimal conditions,the proposed method achieved two linear independent regression equations. When Salmonella choleraesuis concentration increased from 2.54×103 to 6.17×105 CFU/mL,the first regression equation was described as y1=0.1051ln(x)+0.7024(R2=0.7513). With the increase of Salmonella choleraesuis concentration from 6.17×105 to 1.67×107 CFU/mL,the second regression equation was expressed as y2=1.3216ln(x)-15.797(R2=0.9711). The average recoveries of the proposed method for spiked milk samples at Salmonella choleraesuis concentrations ranging from 2.5×103~5.6×106 CFU/mL were 72.16%~103.58% with the relative standard deviations ranging from 7.54%~15.30%. In conclusion,the developed colorimetric ELISA is suitable for the rapid and sensitive quantitation of Salmonella choleraesuis in milk.

Paper-based antibiotic sensor (PAS) relying on colorimetric indirect competitive enzyme-linked immunosorbent assay for quantitative tetracycline and chloramphenicol detection

Antibiotics such as tetracycline (TET) and chloramphenicol (CAP) are widely used, but their abuse leads to antibiotic residue which is harmful to human health. To obtain sensitive and accurate TET and CAP detections, we design the paper-based antibiotic sensor (PAS) relying on the colorimetric indirect competitive ELISA. PAS can be simply fabricated only via wax printing and surface modification, making the cost of single detection rather cheap as ∼0.15 US dollar; and additionally, it can be fabricated and conserved in good stability. Moreover, the PAS supporting smartphone application with the functions of image recording, correction and analysis is also designed for quantitative antibiotic detections. Using a series of standard solutions, it is proved that the PAS could reach the extremely low limits of detection as 0.5 ng/mL and 0.05 ng/mL as well as the wide effective linear detecting ranges as 1∼103 ng/mL and 0.1∼100 ng/mL in the TET and CAP detections, respectively. Moreover, using milk and fish samples, it was also verified that the PAS could quantify the TET and CAP concentrations accurately even in the practical applications.

A reusable PMMA/paper hybrid plug-and-play microfluidic device for an ultrasensitive immunoassay with a wide dynamic range

Conventional colorimetric enzyme-linked immunosorbent assay (ELISA) is a time-consuming laboratory assay that is not very sensitive and consumes a large amount of samples. Herein, the development of a reusable, cost-effective, and eco-friendly poly(methyl methacrylate) (PMMA)/paper hybrid plug-and-play (PnP) device for high-sensitivity immunoassay by analyte enrichment and efficient passing-through washing has been reported. The PMMA device has multiple slots where a pre-patterned paper substrate can be inserted. The sample flows back-and-forth through a low-cost, 3D paper substrate within the PMMA channels, thereby enhancing the amount of analyte adsorbed and dramatically increasing the sensitivity while decreasing the assay time. After the enrichment assay, the paper substrate can simply be pulled out of the device, and the results can be qualitatively viewed with the naked eye or scanned through a simple desktop scanner for quantitative analysis. The paper substrate can be replaced with a new substrate so that the device can be reused. The limits of detection (LODs) of 200 pg/mL for immunoglobulin G (IgG) and 270 pg/mL for hepatitis B surface antigen (HBsAg) were obtained. This IgG assay is at least 10 times more sensitive than commercial ELISA kits. In addition, the PnP ELISA exhibited a significant increase in the linear dynamic range from 3 orders of magnitude in a common paper-based device to a wide range of six orders of magnitude in the PnP hybrid device. This reusable PnP device has great potential for the low-cost yet high-sensitivity detection of infectious diseases, cancers, and other important biomolecules.

Alkaline Phosphatase-Triggered in Situ Formation of Silicon-Containing Nanoparticles for a Fluorometric and Colorimetric Dual-Channel Immunoassay

Enzyme-triggered in situ chromogenic and/or fluorogenic reactions under accessible conditions are significant for developing enzyme activity and related spectroscopic assays. Here, we describe a facile one-pot synthetic strategy to prepare silicon-containing nanoparticles with yellow-green fluorescence and orange-red color by mixing N-[3-(trimethoxysilyl)propyl]ethylenediamine and p-aminophenol (AP) in aqueous solution at a mild temperature. Encouraged by the AP-regulated simple synthetic procedure and the generation of AP from alkaline phosphatase (ALP)-catalyzed hydrolysis of 4-aminophenol phosphate (APP), a fluorometric and colorimetric dual-readout ALP activity assay can be rationally envisioned and developed by employing APP as the substrate. In the wake of the good analytical performance of such ALP activity assay and its successful combination with enzyme-linked immunosorbent assay (ELISA), corresponding fluorometric and colorimetric dual-readout ALP-based ELISA has been constructed for highly sensitive and quantitative determination of human prostate-specific antigen (PSA), the key biomarker of prostate cancer in human serum. The convincing performance in evaluating the PSA level in serologic tests unambiguously reveals the great potential of our proposed fluorometric and colorimetric dual-channel immunoassay in early clinical diagnosis by monitoring disease biomarkers.

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats.

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.

The Effects of Matrine in Combination with Docetaxel on Castration-Resistant (Androgen-Independent) Prostate Cancer.

BackgroundMatrine (MAT) exhibits higher efficacy of chemotherapy when it is combined with other chemotherapeutic drugs; however, the therapeutic efficacy of matrine in combination with docetaxel (DOC) for prostate cancer, or even androgen-independent prostate cancer, remains poorly understood and the underlying molecular mechanisms have not yet been clearly defined. In the present study, we investigated whether matrine combined with docetaxel can strengthen anti-cancer effect.MethodsIn this study, 7 groups were established, including (1) blank control group (cells). (2) 0.1 g/L MAT group, (3) 0.5 g/L MAT group, (4) 0.1 g/L MAT+ 50 μg/L DOC group, (5) 0.5 g/L MAT+ 50 μg/L DOC group, (6) 0.1 g/L MAT+ 100 μg/L DOC group, and (7) 0.5 g/L MAT+ 100 μg/L DOC group. MTS assay was performed to detect the anti-proliferative effects of each group on DU145 and PC-3 cells. At the same time, Transwell assay was performed to detect anti-migrative and anti-invasive effects of each group on DU145 and PC-3 cells. Biochemical colorimetric method and enzyme-linked immunosorbent assay were performed to detect the levels of LDH, IL-1β and IL-18 of each group on DU145 and PC-3 cells. Flow cytometry (FCM) assay was used to do the apoptosis analysis on DU145 and PC-3 cells of each group. At last, Western blot analysis was performed to investigate the expression levels of caspase1 in cells of each group. Statistical analyses were performed with SPSS 17.0 (SPSS Inc, USA) software, and one-way ANOVA and Fisher’s exact test was taken.ResultsMTS assay showed that matrine combined with docetaxel could inhibit both DU145 and PC-3 cells' proliferation in a dose- and time-dependent manner. Transwell assay showed that matrine combined with docetaxel could inhibit both DU145 and PC-3 cells' migration and invasion in a dose- and time-dependent manner. The levels of LDH, IL-1β and IL-18 of matrine combined with docetaxel-treated DU145 and PC-3 cells were significantly increased, compared with the untreated control cells. Flow cytometry, as well as Annexin-V/PI staining, showed a significant and dose-dependent increase in the number of early, as well as late-stage apoptotic cells in both DU145 and PC-3 cells compared with the untreated control cells. Western blot analysis showed that matrine combined with docetaxel treatment led to the expression of caspase1 in both DU145 and PC-3 cells.ConclusionIt may be more effective to use matrine in combination with docetaxel to treat androgen-resistant prostate cancer because matrine can help to affect proliferation, migration, invasion, apoptosis, metabolism, and have anti-inflammation effect on the tumor cells.

Experimental comparison of surface chemistries for biomolecule immobilization on paper-based microfluidic devices

Biomolecules (e.g. proteins and nucleic acids) as target analytes of microfluidic paper-based analytical devices (μPADs) are usually immobilized on a cellulose paper substrate (with intrinsically anionic surface) through physical adsorptions by van der Waals forces and electrostatic interaction thanks to cationic patches on the biomolecule. However, the physical adsorption could lead to weak biomolecule-substrate binding strength and thus low biosensing performance. Benefitting from the abundance of hydroxyl groups on the cellulose paper, chemical modification based on specific surface chemistries is capable of biofunctionalization on the μPADs by providing functional groups for covalent bindings with the target biomolecule. There are many previous reports on chemical modifications of cellulose surface for improvement of biomolecule immobilization. Nevertheless, no study has been performed on experimental evaluation of modification efficiencies of various biofunctionalization methods in the context of biosensing applications. In this paper, we compare five surface chemistries for protein immobilization on μPADs made from pure cellulose paper. For each chemical modification method, surface analyses were first conducted to monitor the surface modification process. Then, paper-based fluorometric experiments and colorimetric enzyme-linked immunosorbent assays (ELISA) were carried out on paper substrates modified by the five surface chemistries to compare their efficiencies of covalent protein immobilization. Finally, a stability experiment was carried out on the five types of surface-modified paper after 30 d storage. It was demonstrated that the potassium periodate (KIO4)-modified cellulose paper has the best performance with 53% increase in the signal output and 59% decrease in background noise of the colorimetric ELISA, and only 13% bioactivity loss after the 30 d storage. The comparison results provide a valuable experimental guideline for selecting the suitable surface chemistry for protein immobilization on μPADs.

A paper-based microfluidic platform with shape-memory-polymer-actuated fluid valves for automated multi-step immunoassays

Smart fluid manipulation with automatically controlled paper valves will enable automated and multi-step immunoassays on paper-based microfluidic devices. In this work, we present an integrated paper-based microfluidic platform with shape-memory polymer (SMP)-actuated fluid valves capable of automated colorimetric enzyme-linked immunosorbent assays (ELISAs). A single-layer microfluidic paper-based analytical device (μPAD) was designed to store all the reagents on the chip, and sequentially transfer reagents to a paper test zone following a specific ELISA protocol through automatic fluidic flow control by the multiple SMP-actuated valves. The actuation of a paper valve was based on the thermally responsive, duel-state shape transformation of a SMP sheet attached to the root of a paper cantilever beam for driving a hydrophilic paper bridge to connect and disconnect two paper channels. A portable colorimetric reader was developed to control the on-chip valve operations, quantify the colorimetric signal output, display the assay result, and wirelessly transmit the data to a smart phone for the application of telemedicine. Reliable operations of the paper valve and the entire μPAD were demonstrated with success rates of 97% and 93%, respectively. A detection mechanism for valve malfunction was designed and confirmed effective to identify any mal-operation of individual valves, thus rendering our platform reliable in real assays. For device calibration, we conducted direct ELISAs of rabbit IgG in phosphate-buffered saline (PBS), and achieved a low limit of detection (LOD) of 27 pM (comparable to that of standard and paper-based ELISAs). In order to demonstrate the clinical application of our multi-step immunoassay platform, we also conducted sandwich ELISAs to quantify the protein level of an inflammatory cytokine, namely tumor necrosis factor (TNF)-α, in surgically injured laryngeal tissues of rats. The protein levels of TNF-α were shown similar between the conventional and μPAD ELISAs.