Photoelectrochemical Detection of Exosomal miRNAs by Combining Target-Programmed Controllable Signal Quenching Engineering.

MicroRNAs extracted from exosomes (exosomal miRNAs) have recently emerged as promising biomarkers for early prognosis and diagnosis. Thus, the development of an effective approach for exosomal miRNA monitoring has triggered extensive attention. Herein, a sensitive photoelectrochemical (PEC) biosensing platform is demonstrated for exosomal miRNA assay via the target miRNA-powered λ-exonuclease for the amplification strategy. The metal-organic framework (MOF)-decorated WO3 nanoflakes heterostructure is constructed and implemented as the photoelectrode. Also, a target exosomal miRNA-activatable programmed release nanocarrier was fabricated, which is responsible for signal control. Hemin that acted as the electron acceptor was prior entrapped into the programmed control release nanocarriers. Once the target exosomal miRNAs-21 was introduced, the as-prepared programmed release nanocarriers were initiated to trigger the release of hemin, which enabled the quenching of the photocurrent. Under the optimized conditions, the level of exosomal miRNAs-21 could be accurately tracked ranging from 1 fM to 0.1 µM with a low detection limit of 0.5 fM. The discoveries illustrate the possibility for the rapid and efficient diagnosis and prognosis prediction of diseases based on the detection of exosomal miRNAs-21 and would provide feasible approaches for the fabrication of an efficient platform for clinical applications.

"Turn-off" sensing probe based on fluorescent gold nanoclusters for the sensitive detection of hemin.

Balanced level of hemin in the body is fundamentally important for normal human organ function. Therefore, environmentally benign, stable, and fluorescent metal nanoclusters (NCs) for selective and sensitive detection of hemin have been investigated and reported. Herein, highly orange red emissive gold NCs are successfully synthesized using glutathione as a reducing and stabilizing agent (GSH-Au NCs). The clusters are characterized using various techniques like Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), UV-vis spectroscopy, and fluorescence spectrometer. The fluorescence intensity of as-synthesized Au NCs strongly quenched upon addition of different concentrations of hemin. The decrease in fluorescence intensity of GSH-Au NCs has been applied for determination of hemin concentration in the linear range from 1 to 25 nM with a low limit of detection (LOD) of 0.43 nM. The method was also successfully applied for quantification of hemin in human serum sample. In view of this reality, the system can be considered as a possible strategy and excellent platform for determination of hemin in various areas of application.

Plasmonic SERS Biosensor Based on Multibranched Gold Nanoparticles Embedded in Polydimethylsiloxane for Quantification of Hematin in Human Erythrocytes.

This work reports a plasmonic surface-enhanced Raman scattering (SERS) biosensor that allows for quantitative analysis of hematin in erythrocytes without the need of separating it from hemoglobin (Hb). The biosensor exploits the tunable localized surface plasmon resonance (LSPR) characteristics of multibranched gold nanoparticles (M-AuNPs) and the strong plasmon coupling between an Au thin film and a flexible substrate consisting of M-AuNPs embedded in polydimethylsiloxane (PDMS) (i.e., M-AuNP-embedded PDMS substrate). In the assay, the hematin (or hematin-containing erythrocyte hemolysate) was deposited on Au film surface and covered with M-AuNP-embedded PDMS. Strong SERS signals were generated under excitation at 785 nm; the signals were sensitive to hematin concentration but not to several common coexisting biological substances. The intensities of the SERS signal (at 1623 cm-1) displayed a wide linear range using hematin concentrations in a range of at least ∼1.5 nM-1.1 µM; the limit of detection (LOD) was ∼0.03 ± 0.01 nM at a signal/noise (S/N) of 3. This assay is simple and sensitive without tedious separation procedures, thereby saving time and enhancing efficiency. This biosensor can be used to determine hematin concentration in human erythrocyte cytosols giving concentrations of ∼18.5 ± 4.5 (by averaging eight samples) and 51.5 ± 6.2 µM (by averaging three samples) for healthy and sickle erythrocytes, respectively, making it a potential application in clinical detection.

Electrochemical determination of artemisinin based on signal inhibition for the reduction of hemin.

A novel electrochemical sensor was constructed for the determination of artemisinin (ART) based on the inhibition of redox for hemin caused by ART. As far as we know, this strategy for ART determination may be proposed for the first time. In this work, untreated multi-walled carbon nanotubes were cast on the glassy carbon electrode (GCE) as conductive carrier. We prepared a bimetallic organic framework named FeGd-MOF and combined it with hemin by a simple physical mixed method. Then, we fabricated the working electrode by layer-by-layer modification and immobilization. The sensor measured by the differential pulse voltammetry (DPV) technique had calibration curves for the determination of ART, which was 0.3-350 µM with the correlation coefficient R2 = 0.9998. Furthermore, the obtained linear range could be practically used in real sample analysis such as dried leaves of Artemisia apiacea. Under the optimized condition, the electrochemical sensor exhibited high sensitivity, good stability, and excellent anti-interference performance. The limit of detection (LOD) for this sensor was 0.17 µM (signal to noise ratio, S/N = 3), which was much lower than that for some other reported electrochemical sensors. The recovery rates were in the range of 99.54-104.34% in real samples, indicating that the sensor had good repetition and high accuracy. Graphical abstract.

Nitrogen-doped MoS2 quantum dots: Facile synthesis and application for the assay of hematin in human blood.

Nitrogen-doped MoS2 quantum dots (N-MoS2 QDs) were synthesized via a facile hydrothermal approach, and exhibited high fluorescence quantum yield (QY, 14.9%), excellent photostability, biocompatibility and water solubility. A novel method with good selectivity and sensitivity was established to assay hematin using N-MoS2 QDs as a fluorescent probe based on inner filter effect (IFE). Fluorescent quenching of N-MoS2 QDs has a fine linear dependence with the concentration of hematin in the range of 0.5-15 µmol/L and a limit of detection of 0.32 µmol/L (S/N = 3). By the detection method, average concentration of hematin in real health human erythrocytes was measured as 22.5 ± 3.9 µmol/L. And, recoveries range varied from 94 to 108% through standard recovery experiment. The N-MoS2 QDs probe shows excellent photostability, low cytotoxicity and anti-interference ability for hematin assay, which may become a promising method for the test of hematin in human blood.

Artesunate-luminol chemiluminescence system for the detection of hemin.

Fabricating simple, accurate and user-friendly diagnostic device for "point of care testing" (POCT) applications is one of the most challenging objectives in the analytical field. Hemin detection is important for drugs monitoring, diagnosis, and forensic latent bloodstain imaging. Herein is developed, luminol chemiluminescence biosensor for hemin detection using artesunate as coreactant. A possible mechanism to account for the chemiluminescence reaction is discussed. Hemin was detected using both photomultiplier tube (PMT) and smartphone as detector. The detection limit for hemin using smartphone as detector is 20 nM, enabling the visual detection of hemin in blood sample with a dilution factor of blood up to 120,000. While PMT detector is used, the system is able to detect hemin down to 0.22 nM. In addition to high sensitivity, this sensing system exhibit high selectivity. It can successfully distinguish bloodstain from other stains while applying the system for point of care testing using smart phone as detector. Moreover, the system can detect artesunate with a linear range from 0.1 nM to 1.0 µM with a limit of detection of 0.078 nM.

Effect of high hydrostatic pressure on the oxidation of washed muscle with added chicken hemoglobin.

The role of chicken hemoglobin in lipid oxidation of washed chicken muscle exposed to high hydrostatic pressure (0, 200, 400 and 600 MPa) was examined. The observed decrease in redness was higher with elevated pressures (5.0 vs 3.7). During storage, redness decreased in samples exposed to 400 and 600 MPa. This decrease was concomitant with the progression of oxidation and the 2.5 fold decrease of soluble heme. An additional experiment was conducted to examine the effect of the hemoglobin mode of addition into pressurized muscle. The exposure at 600 MPa led to washed muscle oxidation even in the absence of hemoglobin, thus indicating that pressure treatments triggered lipid oxidation. However, the presence of native or pressurized hemoglobin into pressurized washed muscle caused more hexanal than the pressurized control without hemoglobin. Overall, results suggest that membrane disruption and the release of hemin are crucial for the onset of oxidation.

Green fluorescent carbon quantum dots as a label-free probe for rapid and sensitive detection of hematin.

Hematin is an oxidized form of heme, and the abnormal levels of hematin in the human body can lead to various inflammatory lesions. Hence, there is still a need to establish a rapid, sensitive and efficient method for hematin detection. Herein, the green fluorescent carbon quantum dots (CQDs) are synthesized by using L-cysteine and hydrogen peroxide as precursors. The synthesized CQDs exhibit some fascinating characters including excellent water solubility, high fluorescence quantum yield, and good stability in a broad pH range of 7.0-11.0 and high ionic strength solution. Excitingly, the fluorescence of CQDs can be rapidly and selectively quenched by hematin via the inner filter effect. Moreover, the detection of hematin by the CQDs fluorescent probe shows a good linearity in the concentration range of 0.5-30 µM with a minimum detection limit of 0.1 µM. Finally, the proposed approach is successfully applied to detect hematin in human blood samples.

Proton-controlled synthesis of red-emitting carbon dots and application for hematin detection in human erythrocytes.

The Red-emitting nitrogen-doped carbon dots (N-CDs) are synthesized using o-phenylenediamine by a one-step method, and can serve as a fluorescent probe for "turn off" detection of hematin in human red cells. The red-emitting N-CDs can be obtained only in acidic conditions and the emission of the red-emitting N-CDs is pH-dependent, indicating proton-controlled synthesis and emission. The red-emitting N-CDs are 2.7 nm in mean size and have a uniform dispersion and exhibit a high quantum yield (12.8%) and great optical properties. The developed sensing system for hematin displays a linear response from 0.4 to 32 µM with a detection limit of 0.18 µM. Importantly, this fluorescent probe demonstrates a good potential practicability for the quantitative detection of hematin in complex matrixes. Graphical abstract ᅟ.

A systematic review of methods to measure menstrual blood loss.

BACKGROUND: Since the publication over 50 years ago of the alkaline hematin method for quantifying menstrual blood loss (MBL) many new approaches have been developed to assess MBL. The aim of this systematic review is to determine for methods of measuring MBL: ability to distinguish between normal and heavy menstrual bleeding (HMB); practicalities and limitations in the research setting; and suitability for diagnosing HMB in routine clinical practice. METHODS: Embase®™, MEDLINE®, and ClinicalTrials.gov were screened for studies on the development/validation of MBL assessment methods in women with self-perceived HMB, actual HMB or uterine fibroids, or patients undergoing treatment for HMB. Studies using simulated menstrual fluid and those that included women with normal MBL as controls were also eligible for inclusion. Extracted data included study population, results of validation, and advantages/disadvantages of the technique. RESULTS: Seventy-one studies fulfilled the inclusion criteria. The sensitivity and/or specificity of diagnosing HMB were calculated in 16 studies of methods involving self-perception of MBL (11 pictorial), and in one analysis of the menstrual-fluid-loss (MFL) method; in 13 of these studies the comparator was the gold standard alkaline hematin technique. Sensitivity and specificity values by method were, respectively: MFL model, 89, 98%; pictorial blood loss assessment chart (PBAC), 58-99%, 7.5-89%; menstrual pictogram, 82-96%, 88-94%; models/questionnaires, 59-87%, 62-86%, and complaint of HMB, 74, 74%. The power of methods to identify HMB was also assessed using other analyses such as comparison of average measurements: statistical significance was reported for the PBAC, MFL, subjective complaint, and six questionnaires. In addition, PBAC scores, menstrual pictogram volumes, MFL, pad/tampon count, iron loss, and output from three questionnaires correlated significantly with values from a reference method in at least one study. In general, pictorial methods have been more comprehensively validated than questionnaires and models. CONCLUSIONS: Every method to assess MBL has limitations. Pictorial methods strike a good balance between ease of use and validated accuracy of MBL determination, and could complement assessment of HMB using quality of life (QoL) in the clinical and research setting. TRIAL REGISTRATION: PRISMA registration number: CRD42016032956 .

Inner filter with carbon quantum dots: A selective sensing platform for detection of hematin in human red cells.

Hematin plays a crucial role in various physiological functions, and the determination of hematin in complex biological matrixes is a significant but difficult issue. Considering the unique photophysical/photochemical properties of carbon quantum dots (CQDs) prepared with p-aminobenzoic acid (PABA) and ethanol, a new strategy for the design of fluorescent probes for hematin has been achieved. The proposed sensor array is fabricated based on the inner filter effect (IFE) between hematin and CQDs with phenomenon of selective fluorescence quenching of CQDs which results from the strong absorption of the excitation and emission spectrum of CQDs by hematin. The fluorescence quenching of CQDs is closely related to the amount of hematin and there is a good linear relationship over the range of 0.5-10µM with a detection limit of 0.25µM. What's more, the fluorescence assay has been successfully applied for hematin sensing in healthy human red cells showing this sensing assay has a great potential prospect for detection of hematin in the complex matrixes.

Artemisinin-Luminol Chemiluminescence for Forensic Bloodstain Detection Using a Smart Phone as a Detector.

Forensic luminol chemiluminescence test is one of the most sensitive and popular methods for the determination of latent bloodstains. It mainly uses hydrogen peroxide or sodium perborate as coreactants. The easy decomposition of hydrogen peroxide and sodium perborate in the presence of many ions significantly affects the selectivity. Artemisinin is a natural peroxide that is quite stable in the presence of common ions. In the present study, artemisinin has been exploited for the forensic bloodstain chemiluminescence detection for the first time. Using smart phone as cost-effective portable detector, the visual detection of bloodstains has been achieved with a dilution factor of blood up to 100 000. Moreover, this system shows excellent selectivity against many common species. It can well differentiate bloodstains from other stains, such as coffee, brown sugar, and black tea. Both favorable sensitivity and selectivity makes the present method promising in forensic detection.

A Label-free, Highly Sensitive and Selective Detection of Hemin Based on the Competition between Hemin and Protoporphyrin IX Binding to G-Quadruplexes.

Herein is reported a simple and label-free fluorescent detection method for hemin based on using protoporphyrin IX (PPIX) as a fluorescent signal reporter. PPIX emits weak fluorescence in an aqueous solution. When PPIX binds to G-quadruplexes, the fluorescence intensity of PPIX is greatly increased. While in the presence of target hemin, hemin competes with PPIX toward G-quadruplexes because its affinity to G-quadruplexes is higher than that of PPIX. With the formation of the hemin-G-quadruplex complex, PPIX is released to the solution from the G-quadruplex accompanied by quenching of the fluorescence of the system. This fluorescence change of the system can be used to monitor hemin with a low detection limit of 36 nM. In addition, the possible binding sites for PPIX binding to the G-quadruplex are discussed based on competition between hemin and PPIX. What is more, this method might pave the way for applying G-quadruplexes and PPIX to more sensing systems.

Highly Selective Fluorescence Determination of the Hematin Level in Human Erythrocytes with No Need for Separation from Bulk Hemoglobin.

Hematin-induced fluorescence quenching of boron-doped graphene quantum dots (BGQDs) allows for determination of hematin concentration in human erythrocytes with no need for separating hematin from hemoglobin before performing the assay. The BGQDs are made by oxidizing a graphite anode by holding the voltage between a graphite rod and a Pt cathode at 3 V for 2 h in an aqueous borax solution at pH 7; then, the borate solution was filtered with BGQDs, and the borate was dialyzed from the filtrate, leaving a solution of BGQDs in water. The fluorescence intensity of BGQDs is measurable in real time, and its quenching is very sensitive to the concentration of hematin in the system but not to other coexisting biological substances. The analytical signal is defined as ΔF = 1 - F/F0, where F0 and F are the fluorescence intensities of the BGQDs before and after interaction with hematin, respectively. There is a good linear relationship between ΔF and hematin concentration, ranging from 0.01 to 0.92 µM, with the limit of detection (LOD) being ∼0.005 ± 0.001 µM at a signal-to-noise ratio of 3. This new method is sensitive, label-free, simple, and inexpensive, and many tedious procedures related to sample separation and preparation can be omitted, implying that this method has potential for applications in clinical examinations and disease diagnoses. For example, the determination of the hematin levels in two kind of red blood cell samples, healthy human and sickle cell erythrocytes, gives average concentrations of hematin of ∼(23.1 ± 4.9) µM (average of five samples) for healthy red cell cytosols and ∼(52.5 ± 9.5) µM (average of two samples) for sickle red cell cytosols.

Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

OBJECTIVE: The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). RESULTS: Cell biomass and biomass yield of L. lactis grown with 4 µg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. CONCLUSION: The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

In situ surface-enhanced Raman spectroelectrochemical analysis system with a hemin modified nanostructured gold surface.

An integrated surface-enhanced Raman scattering (SERS) spectroelectrochemical (SEC) analysis system is presented that combines a small volume microfluidic sample chamber (<100 µL) with a compact three-electrode configuration for in situ surface-enhanced Raman spectroelectrochemistry. The SEC system includes a nanostructured Au surface that serves dual roles as the electrochemical working electrode (WE) and SERS substrate, a microfabricated Pt counter electrode (CE), and an external Ag/AgCl reference electrode (RE). The nanostructured Au WE enables highly sensitive in situ SERS spectroscopy through large and reproducible SERS enhancements, which eliminates the need for resonant wavelength matching of the laser excitation source with the electronic absorption of the target molecule. The new SEC analysis system has the merits of wide applicability to target molecules, small sample volume, and a low detection limit. We demonstrate in situ SERS spectroelectrochemistry measurements of the metalloporphyrin hemin showing shifts of the iron oxidation marker band ν4 with the nanostructured Au working electrode under precise potential control.

"Meat juice" is not a homogeneous serological matrix.

Meat juice samples are used in serological assays to monitor infectious diseases within the food chain. However, evidence of inferior sensitivity, presumably due to low levels of antibodies in the meat juice compared to serum, has been presented, and it has been suggested that adjusting the dilution factor of meat juice in proportion to its blood content could improve sensitivity. In the present study, the agreement between Toxoplasma gondii-specific immunoglobulin G (IgG) levels in meat juice and serum was evaluated, and whether the level of immunoglobulins in meat juice was dependent on its blood content. Serum and meat juice from diaphragm, heart, tongue, Musculus triceps brachii and M. semitendinosus were collected from 20 pigs experimentally infected with T. gondii. Analysis of total IgG, heme-containing proteins (hematin), and hemoglobin (Hb) revealed significant differences between samples from different muscles, with the highest levels in samples from heart and tongue, and the lowest in samples from leg muscles. Comparison of T. gondii-specific antibody titers in meat juice and serum revealed a strong positive correlation for meat juice from heart (rs=0.87; p<0.001), while it was lower for M. semitendinosus (rs=0.71; p<0.001) and diaphragm (rs=0.54; p=0.02). Meanwhile, the correlation between total IgG and T. gondii titer ratio (meat juice/serum) was highest in diaphragm (rs=0.77; p<0.001) followed by M. semitendinosus (rs=0.64; p=0.005) and heart (rs=0.50; p=0.051). The correlation between Hb and T. gondii titer ratio was only significant for diaphragm (rs=0.65; p=0.008), and for hematin no significant correlation was recorded. In conclusion, the specific IgG titers in meat juice appeared to depend on the total IgG level, but the correlation to blood (Hb or hematin) was poor. Importantly, large significant differences in total IgG levels as well as in specific antibody titers were recorded, depending on the muscle the meat juice had been extracted from.

A carboxylated graphene and aptamer nanocomposite-based aptasensor for sensitive and specific detection of hemin.

A unique nanocomposite was crafted by grafting hemin-binding-aptamer (HBA) onto carboxylated graphene (COO-GR). Infrared spectroscopy, Raman spectroscopy and diffuse reflectance spectra suggested that -NHCO- covalent bonds were formed between HBA and COO-GR. The resulting COO-GR/HBA functionalized electrode was used as a novel label-free biosensor. The square wave voltammetry was employed to realize the selective and specific detection of hemin. The obtained aptasensor possessed excellent performance with a detection limit of 0.64 nmol L(-1) (S/N=3) and a linear range from 1 to 150 nmol L(-1). Moreover, COO-GR was shown to be a promising candidate in making aptasensors, carrying advantages over graphene in terms of the simplicity of sensor preparation and the reduction of background noise.

Determination of selected protoporphyrins in parma ham with use of 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin as a surrogate standard in the recovery study.

A high-performance liquid chromatographic method for the determination of hemin, protoporphyrin IX (PPIX), and zinc(II)protoporphyrin IX (Zn(II)PPIX) in Parma ham was developed. The detection was done by means of a universal DAD-detector, whereby quantification of the three naturally occurring protoporphyrins was carried out at lambda = 414 nm, i.e., very close to the respective maxima of their Soret bands. The extraction thereof from the meat matrix was done by a mixture of acetone and chloroacetic acid (100 mL + 0.2 g). Usage of 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (THPP) as a surrogate standard and its detection fixed at lambda = 444 nm, allowed to obtain accurate (ca. 96%) recovery results. Established concentrations of hemin, Zn(II)PPIX, and PPIX in the Parma ham samples were 15.97, 19.96 and 1.52 µg g(-1), respectively.

Pharmacokinetics study of hemin in rats by applying (58)Fe-extrinsically labeling techniques in combination with ICP-MS method.

Iron is a challenging element due to its high background in various matrixes including blood, tissues even in the air and it is urgent to develop a method for the accurate determination of iron in bio-samples. After optimization of mass spectrometric conditions using collision cell technology and compensating for interference using a mathematical correction equation, an inductively coupled plasma mass spectrometry (ICP-MS) method for the quantitative determination of (58)Fe originating from hemin extrinsically labeled avoiding endogenous interference was developed. After a single step of dilution, analysis of each sample was completed within 1.5min. The assay was linear in the concentration range from 0.005 to 1.0µg/ml. The precisions and accuracies determined within three consecutive days were in acceptable limits and there was no significant matrix effect. The optimized method was successfully applied to a pharmacokinetic study of (58)Fe originating from hemin extrinsically labeled and iron absorption measured in rats was 1.07%. Those indicated that extrinsically label techniques in combination with ICP-MS will become a new tool for the analysis of iron preparations and other endogenous substances.