We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein ...delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595
nm) can be identified and corrected by recording absorption spectra in the region of 350–850
mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595
nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595
nm by measuring the sample absorbance at 850
nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595
nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.
A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the ...proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5–103.3%.
Graphical abstract
A fluorometry method for thrombin detection using magnetic nanoparticles and
enzyme-free hybridization chain reaction
Surface-enhanced Raman spectroscopy (SERS) is a promising platform for simple, rapid, and economical protein quantitation and analysis and can achieve much lower detection limits for the ...ultrasensitive detection of proteins and a much wider linear concentration range for quantitative analysis than other methods can. In addition, SERS can provide a large amount of fingerprint information for the individual components of a mixture through SERS effects, which are sensitive and selective for different types of proteins and protein mixtures. In general, the occurrence and development of diseases are accompanied by changes in the content or structure of biomarkers (disease-related proteins). Here, we provide an overview of the SERS technique and its applications to disease-related protein determination. Different diseases, such as Alzheimer's disease (AD), cardiac muscle tissue injury, and multicancer, are discussed and exhibit potential utility in biomarker detection and diagnosis. SERS opens a new path to the early diagnosis of critical diseases, which will effectively reduce human suffering and mortality.
Display omitted
A new accurate spectrophotometric method for protein determination on nanoparticles is described. The method is based on the Coomassie blue dye that binds to the basic and aromatic ...amino acid residues of proteins, especially arginine and lysine. A known amount of reagent dye was mixed with a variety of protein-loaded nanoparticles. Thereafter the unconjugated reagent was mixed with excess protein (bovine serum albumin) and titrated. In this method, the reacted dye on the protein coating of nanoparticle is directly determined, in opposite to the conventional method, in which the conjugated protein is determined as the difference between the non-conjugated protein found in the supernatant after centrifugation, and the total amount of protein originally used. This method is able to measure amounts of coated protein lower than 1 ppm.
•Simple and accurate method especially adapted for protein-coated nanoparticles.•The amino acid residues of protein in the nanoparticle surface react with Coomassie brilliant blue dye.•The unreacted dye is titrated with an excess of a standard protein.
Down syndrome (DS) may be considered a genetic form of Alzheimer's disease (AD) due to universal development of AD neuropathology, but diagnosis and treatment trials are hampered by a lack of ...reliable blood biomarkers. A potential biomarker is neurofilament light (NF-L), due to its association with axonal damage in neurodegenerative conditions.
We measured blood NF-L concentrations in 100 adults with DS using Simoa NF-light® assays, and we examined relationships with age as well as cross-sectional and longitudinal dementia diagnosis.
NF-L concentrations increased with age (Spearman's rho = 0.789, p < 0.001), with a steep increase after age 40, and they were predictive of dementia status (p = 0.022 adjusting for age, sex, and APOE4), but they showed no relationship with long-standing epilepsy or premorbid ability. Baseline NF-L concentrations were associated with longitudinal dementia status.
NF-L is a biomarker for neurodegeneration in DS with potential for use in future clinical trials to prevent or delay dementia.
The determination of proteins is very important in industry, medicine, analytical chemistry, and biotechnology. A novel colorimetric method using magnetic microparticles as a carrier of immobilized ...microbial protease on surface was developed and smartphone camera was employed for detection. Folin and Ciocalteu's phenol reagent was selected as the chromogen and casein from bovine milk was used as the substrate. Casein was cleaved by immobilized enzyme to tyrosine, which together with chromogen spontaneously generated a blue color measurable by a spectrophotometer or photographic detection by a smartphone camera. The photographs were processed by a computer and selected points were transformed into RGB numeric values. A reference spectrophotometric method provided results that provide a Michaelis-Menten dependence with the Michaelis-Menten constant equal to 0.11 mmol/l and a limit of detection equal to 41 µg/ml. The novel method using spectrophotometric detection shows the same Michaelis-Menten dependence with a Michaelis-Menten constant equal to 0.11 mmol/l with a limit of detection of 4.27 µg/ml. The RGB method using a smartphone for the determination of proteins was demonstrated to be an alternative to the standard spectrophotometer. The limit of detection was 242 µg/ml, which is sufficient for protein determination in real samples. Easy processing, good analytical results, and low demands on equipment make this method a suitable tool for a wide range of applications in industry, analytical chemistry, medicine, biotechnology, and biosensors.
A new perspective on the relevant problem—creating simple, rapid, and efficient protein sensors based on microstructured optical fibers using a simple homogeneous analysis format—was proposed. ...Commercially available long-period grating hollow core microstructured optical fibers (LPG HCMOF) were used to determine bovine serum albumin (BSA) and albumin from chicken eggs (OVA) in binary mixtures as well as immunoglobulin G (IgG) in the presence of BSA and OVA. LPG HCMOF transmission spectra allowed the detection of both BSA and OVA up to 10 mg/mL with LOD as low as 0.1 and 0.8 μg/mL, respectively. Partial least squares regression (PLS) was utilized for modeling of LPG HCMOF spectral data and quantitative analysis of BSA, OVA, total protein, and IgG in binary and ternary mixtures. Rather high coefficients of determination (
R
2
) and low root mean square error for the calibration (RMSEC) (15%) and prediction (RMSEP) (20%) were obtained for all PLS models. The proposed approach was tested in the analysis of BSA in spiked horse blood hemolyzed (HBH). The results demonstrated the functionality of the proposed approach and offered the opportunity for the creation of a wide range of sensors for protein determination in complex mixtures.
Graphical abstract
Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical methods employed. The different extraction procedures (exposure to water, grinding, ...etc.), protein precipitation using different amounts of 25% trichloroacetic acid and quantification of protein by two different methods and using two protein standards were evaluated. All procedures were tested using freeze-dried samples of three macroalgae: Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocol for protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure water for 12 h, followed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should be done with 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in this study was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima, Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae (Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Comparison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry's method should be used instead of Bradford's using bovine serum albumin (BSA) as protein standard instead of casein. This may be related to the reactivity of the protein standards and the greater similarity in the amino acid composition of BSA and algae. The current results should contribute to more accurate protein determinations in marine algae.
This paper proposes a methodology for the classification and determination of total protein in milk powder using near infrared reflectance spectrometry (NIRS) and variable selection. Two brands of ...milk powder were acquired from three Brazilian cities (Natal-RN, Salvador-BA and Rio de Janeiro-RJ). The protein content of 38 samples was determined by the Kjeldahl method and NIRS analysis. Principal component regression (PCR) and partial least squares (PLS) multivariate calibrations were used to predict the total protein. Soft independent modeling of class analogy (SIMCA) was also used for full-spectrum classification, resulting in almost 100% classification accuracy, regardless of the significance level adopted for the
F-test. Using this strategy, it was feasible to classify powder milk rapidly and nondestructively without the need for various analytical determinations. Concerning the multivariate calibration models, the results show that PCR, PLS and MLR-SPA models are good for predicting total protein in powder milk; the respective root mean square errors of prediction (RMSEP) were 0.28 (PCR), 0.25 (PLS), 0.11
wt% (MLR-SPA) with an average sample protein content of 8.1
wt%. The results obtained in this investigation suggest that the proposed methodology is a promising alternative for the determination of total protein in milk powder.
PDF
