RESUMEN
Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples.
Asunto(s)
Péptido Natriurético Encefálico , Fragmentos de Péptidos , Humanos , Péptido Natriurético Encefálico/sangre , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/sangre , Pruebas en el Punto de Atención , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Diseño de EquipoRESUMEN
A facile strategy was introduced for room-temperature controllable synthesis of hierarchically flower-like hollow COFs (FHF-COFs). Furthermore, the universality for synthesis of the HFH-COFs was validated by altering the building units. Inspired by the unique morphology, extremely large surface area and good chemical stability, HFH-COFs could serve as an attractive adsorption probe by loading with gold nanoparticles and be applied to enrichment of brain natriuretic peptide from human serum. This work opens up a whole new approach for controllable synthesis of the HFH-COFs at room temperature and expands the application of COFs as a promising enrichment probe for complex biological samples.
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Estructuras Metalorgánicas/química , Péptido Natriurético Encefálico/aislamiento & purificación , Adsorción , Oro/química , Humanos , Límite de Detección , Nanopartículas del Metal/química , Estructuras Metalorgánicas/síntesis química , Péptido Natriurético Encefálico/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Heart failure is a class of cardiovascular diseases that remains the number one cause of death worldwide with a substantial economic burden of around $18 billion incurred by the healthcare sector in 2017 due to heart failure hospitalization and disease management. Although several laboratory tests have been used for early detection of heart failure, these traditional diagnostic methods still fail to effectively guide clinical decisions, prognosis, and therapy in a timely and cost-effective manner. Recent advances in the design and development of biosensors coupled with the discovery of new clinically relevant cardiac biomarkers are paving the way for breakthroughs in heart failure management. Natriuretic neurohormone peptides, B-type natriuretic peptide (BNP) and N-terminal prohormone of BNP (NT-proBNP), are among the most promising biomarkers for clinical use. Remarkably, they result in an increased diagnostic accuracy of around 80% owing to the strong correlation between their circulating concentrations and different heart failure events. The latter has encouraged research towards developing and optimizing BNP biosensors for rapid and highly sensitive detection in the scope of point-of-care testing. This review sheds light on the advances in BNP and NT-proBNP sensing technologies for point-of-care (POC) applications and highlights the challenges of potential integration of these technologies in the clinic. Optical and electrochemical immunosensors are currently used for BNP sensing. The performance metrics of these biosensors-expressed in terms of sensitivity, selectivity, reproducibility, and other criteria-are compared to those of traditional diagnostic techniques, and the clinical applicability of these biosensors is assessed for their potential integration in point-of-care diagnostic platforms.
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Técnicas Biosensibles , Insuficiencia Cardíaca/diagnóstico , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Biomarcadores/análisis , Humanos , Péptidos Natriuréticos/aislamiento & purificación , Sistemas de Atención de PuntoRESUMEN
Surface-plasmon-resonance (SPR) is a quantum-electromagnetic phenomenon arising from the interaction of light with free electrons at a metal-dielectric interface. At a specific angle/wavelength of light, the photon's energy is transferred to excite the oscillation of the free electrons on the surface. A change in the refractive-index (RI) may occur, which is influenced by the analyte concentration in the medium in close contact with the metal surface. SPR has been widely used for the detection of gaseous, liquid, or solid samples. In this study, a functionalized specific SPR chip was designed and used in a novel point-of-care SPR module (PhotonicSys SPR H5) for the detection of the stroke biomarkers NT-proBNP and S100ß. These biomarkers have proven to be good for stroke diagnosis, with sensitivity and specificity of >85%. Specific detection was done by binding a biomolecular-recognizing antibody onto the Au SPR-chip. Detection was tested in water and plasma samples. NT-proBNP and S100ß were detected in a range of concentrations for stroke, from 0.1 ng/mL to 10 ng/mL. The RI of the blank plasma samples was 1.362412, and the lowest concentration tested for both biomarkers showed a prominent shift in the RI signal (0.25 ng/mL NT-proBNP (1.364215) and S100ß (1.364024)). The sensor demonstrated a clinically relevant limit-of-detection of less than ng/mL.
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Técnicas Biosensibles , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Subunidad beta de la Proteína de Unión al Calcio S100/aislamiento & purificación , Accidente Cerebrovascular/diagnóstico , Anticuerpos/química , Anticuerpos/inmunología , Biomarcadores/química , Oro/química , Humanos , Péptido Natriurético Encefálico/química , Fragmentos de Péptidos/química , Sistemas de Atención de Punto , Subunidad beta de la Proteína de Unión al Calcio S100/química , Resonancia por Plasmón de SuperficieRESUMEN
A sandwich-type photoelectrochemical (PEC) immunosensing platform was designed for detection of amino-terminal pro-B-type natriuretic peptide (NT-pro BNP). Thereinto, flower-like Bi2WO6/Ag2S nanoparticles (F-Bi2WO6/Ag2S) were employed as photoelectrochemical matrix, and graphene oxide and polydopamine composite (GO/PDA) were prepared as signal labels. In this proposal, Ag2S was in-situ growth on the surface of F-Bi2WO6 modified with thioglycolic acid (TGA). Specially, a cascade-like band-edge level between F-Bi2WO6 and Ag2S effectively improved the photocurrent conversion efficiency and enhanced the photocurrent response. Then, the conjugated GO/PDA aimed to further amplify signal because PDA as electron donor could sweep the holes and inhibit the recombination of photogenerated electron-hole pairs, while GO owned brilliant conductivity speeding up the electrons transfer. The photocurrent increased with the amount of GO/PDA conjugates which had positive correlation with the NT-pro BNP. Under optimal experimental conditions, the proposed sandwich-type PEC immunosensor presented a desirable linear relationship ranged from 0.1â¯pg/mL to 100â¯ng/mL for NT-pro BNP with the detection limit of 0.03â¯pg/mL (S/Nâ¯=â¯3). The prepared PEC immunosensor exhibited high stability and selectivity, which offered an innovative idea for the detection of other biomolecules.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Inmunoensayo , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Compuestos de Cadmio/química , Grafito/química , Humanos , Indoles/química , Nanocompuestos/química , Péptido Natriurético Encefálico/química , Fragmentos de Péptidos/química , Polímeros/químicaRESUMEN
In this work, by using amorphous bimetallic sulfides of CoSnSxPd as signal amplifier and magnetic material of Fe3O4 @PPyAu as the substrate, we fabricated an electrochemical immunoassay for Nterminal prohormone of brain natriuretic peptide (NTpro BNP). CoSnSx was used as the signal amplifier of the immunosensor for the first time, which was synthesized by fast stoichiometric co-precipitation method. The binary sulfide was identified as an amorphous structure and performed apparent electrochemical behavior. The excellent performance of signal marker was induced by the synergistic effect between SnS2 and cobalt, as well as the loaded Pd nanoparticles (NPs). Both CoSnSx and Pd NPs presented excellent electrocatalytic activity toward H2O2 oxidation. Moreover, the magnetic nanocomposite of Fe3O4 @PPyAu was firmly immobilized on the magnetic glassy carbon electrode (MGCE) by the magnetic force. The magnetic substrate not only performed good electron conductivity, but also improved the stability of the fabricated electrochemical immunosensor. The developed immunosensor for the NTpro BNP detection exhibited a wide linear response (0.1â¯pg/mL to 50â¯ng/mL), and low detection limit of 31.5â¯fg/mL. In addition, the immunosensor held an excellent analysis capability and broad fascinating application for the detection of other biomarkers in medical diagnosis and treatment.
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Técnicas Biosensibles , Técnicas Electroquímicas , Nanopartículas del Metal/química , Péptido Natriurético Encefálico/aislamiento & purificación , Carbono/química , Compuestos Férricos/química , Oro/química , Grafito/química , Humanos , Peróxido de Hidrógeno/química , Péptido Natriurético Encefálico/química , Paladio/química , Sulfuros/química , Compuestos de Estaño/químicaRESUMEN
Cardiovascular diseases (CVD) remain the leading cause of death within industrialized nations as well as an increasing cause of mortality and morbidity in many developing countries. Smoking, alcohol consumption and increased level of blood cholesterol are the main CVD risk factors. Other factors, such as the prevalence of overweight/obesity and diabetes, have increased considerably in recent decades and are indirect causes of CVD. Among CVDs, the acute coronary syndrome (ACS) represents the most common cause of emergency hospital admission. Since the prognosis of ACS is directly associated with timely initiation of revascularization, missed, misdiagnosis or late diagnosis have unfavorable medical implications. Early ACS diagnosis can reduce complications and risk of recurrence, finally decreasing the economic burden posed on the health care system as a whole. To decrease the risk of ACS and related CVDs and to reduce associated costs to healthcare systems, a fast management of patients with chest pain has become crucial and urgent. Despite great efforts, biochemical diagnostic approaches of CVDs remain difficult and controversial medical challenges as cardiac biomarkers should be rapidly released into the blood at the time of ischemia and persistent for a sufficient length of time to allow diagnostics, with tests that should be rapid, easy to perform and relatively inexpensive. Early biomarker assessments have involved testing for the total enzyme activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK), which cardiac troponins being the main accepted biomarkers for diagnosing myocardial injury and acute myocardial infarction (AMI). To allow rapid diagnosis, it is necessary to replace the traditional biochemical assays by cardiac biosensor platforms. Among the numerous of possibilities existing today, electrochemical biosensors are important players as they have many of the required characteristics for point-of-care tests. Electrochemical based cardiac biosensors are highly adapted for monitoring the onset and progress of cardiovascular diseases in a fast and accurate manner, while being cheap and scalable devices. This review outlines the state of the art in the development of cardiac electrochemical sensors for the detection of different cardiac biomarkers ranging from troponin to BNP, N-terminal proBNP, and others.
Asunto(s)
Síndrome Coronario Agudo/sangre , Biomarcadores/sangre , Técnicas Biosensibles , Enfermedades Cardiovasculares/sangre , Humanos , Péptido Natriurético Encefálico/sangre , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/aislamiento & purificación , Pronóstico , Factores de Riesgo , Troponina/sangre , Troponina/aislamiento & purificaciónRESUMEN
A sensitive dual mode competitive electrochemical immunosensor for the detection of B-type natriuretic peptide (BNP) was successfully fabricated, which based on differential pulse voltammetry (DPV) and amperometric i-t curve response modes. Polyaniline (PAN) and tin dioxide (SnO2) were loaded on graphene sheets (GS), which could effectively promote the electron transfer process, thereby amplifying the current signal and increasing the sensitivity of the immunosensor. To promote biocompatibility, gold nanoparticles (Au) were incorporated on GS/SnO2/PAN (GS/SnO2/PAN-Au). GS/SnO2/PAN-Au complex was gotten to act as the platform which could provide a clearly DPV signals. N-doped carbon nanotubes (N-CNTs) embellished by ZnCo2O4 quantum dots (ZnCo2O4/N-CNTs) with excellent catalytic properties for the reduction of H2O2 was gotten to act as the label of the antibody-BNP (Ab), providing an obviously current signal through amperometric i-t curve method. A large quantity of BNP could be stable loaded in the modified electrode via GS/SnO2/PAN-Au with excellent electrical conductivity and good biocompatibility, which could compete with target-BNP to combine Ab that labelled by ZnCo2O4/N-CNTs. Under the optimum conditions, the immunosensor exhibited remarkable analytical performance of a linear range from 0.01â¯pg/mL to 1â¯ng/mL with a detection limit of 3.4 fg/mL for quantitative detection of BNP (S/Nâ¯=â¯3). This method was able to become a universal strategy for other biological detection.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Inmunoensayo , Péptido Natriurético Encefálico/aislamiento & purificación , Compuestos de Anilina/química , Cobalto/química , Oro/química , Grafito/química , Humanos , Nanopartículas del Metal/química , Nanotubos de Carbono/química , Péptido Natriurético Encefálico/química , Compuestos de Estaño/química , Zinc/químicaRESUMEN
A novel electrochemiluminescence (ECL) signal-off strategy based on CuS in situ grown on reduced graphene oxide (CuS-rGO) quenching luminol/H2O2 system was firstly proposed. Luminol was grafted on the surface of Au@Fe3O4-Cu3(PO4)2 nanoflowers (Luminol-Au@Fe3O4-Cu3(PO4)2) which exhibited excellent catalytic effect towards the reduction of H2O2 to enhance the ECL intensity of luminol. Cu3(PO4)2 nanoflowers showed large surface area which can immobilize more Fe3O4 and Au nanoparticles. The quenching mechanism of CuS-rGO was due to ECL resonance energy transfer (RET). The spectral overlap between fluorescence spectrum of Luminol-Au@Fe3O4-Cu3(PO4)2 and UV-vis absorption spectrum of CuS-rGO revealed that resonance energy transfer was possible. Au nanoparticles were immobilized on the surface of CuS-rGO to capture secondary antibodies. After a sandwich-type immunoreaction, a remarkable decrease of ECL signal was observed. Under the optimal conditions, the immunosensor showed excellent performance for N-terminal pro-brain natriuretic peptide (NT-proBNP) detection with a wide detection range from 0.5â¯pgâ¯mL-1 to 20â¯ngâ¯mL-1 and a low detection limit of 0.12â¯pgâ¯mL-1 (S/Nâ¯=â¯3). The prepared NT-proBNP immunosensor displayed high sensitivity, excellent stability and good specificity.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Grafito/química , Péptido Natriurético Encefálico/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Cobre/química , Glucosa Oxidasa/química , Oro/química , Humanos , Peróxido de Hidrógeno/química , Límite de Detección , Luminol/química , Nanopartículas del Metal/química , Péptido Natriurético Encefálico/química , Fragmentos de Péptidos/químicaRESUMEN
A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy)3] doped silica nanoparticles (SiO2@Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H2O2 for efficiently quenching the ECL emission of SiO2@Ir. Typically, the SiO2@Ir not only increased the loading amount of Ir(ppy)3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H2O2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO2@Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Nanopartículas del Metal/química , Péptido Natriurético Encefálico/aislamiento & purificación , Glucosa Oxidasa/química , Glucosa Oxidasa/inmunología , Humanos , Peróxido de Hidrógeno/química , Iridio/química , Límite de Detección , Péptido Natriurético Encefálico/química , Péptido Natriurético Encefálico/inmunología , Dióxido de Silicio/químicaRESUMEN
BACKGROUND: B-type natriuretic peptide (BNP) is a 32 amino acid cardiac hormone routinely measured by immunoassays to diagnose heart failure. While it is reported that immunoassay results can vary up to 45%, no attempt of standardization and/or harmonization through the development of certified reference materials (CRMs) or reference measurement procedures (RMPs) has yet been carried out. METHODS: B-type natriuretic peptide primary calibrator was quantified traceably to the International System of Units (SI) by both amino acid analysis and tryptic digestion. A method for the stabilization of BNP in plasma followed by protein precipitation, solid phase extraction (SPE) and liquid chromatography (LC) mass spectrometry (MS) was then developed and validated for the quantification of BNP at clinically relevant concentrations (15-150 fmol/g). RESULTS: The candidate reference method was applied to the quantification of BNP in a number of samples from the UK NEQAS Cardiac Markers Scheme to demonstrate its applicability to generate reference values and to preliminary evaluate the commutability of a potential CRM. The results from the reference method were consistently lower than the immunoassay results and discrepancy between the immunoassays was observed confirming previous data. CONCLUSIONS: The application of the liquid chromatography-mass spectrometry (LC-MS) method to the UK NEQAS samples and the correlation of the results with the immunoassay results shows the potential of the method to support external quality assessment schemes, to improve understanding of the bias of the assays and to establish RMPs for BNP measurements. Furthermore, the method has the potential to be multiplexed for monitoring circulating truncated forms of BNP.
Asunto(s)
Péptido Natriurético Encefálico/sangre , Biomarcadores/sangre , Cromatografía Liquida , Humanos , Inmunoensayo , Espectrometría de Masas , Péptido Natriurético Encefálico/aislamiento & purificación , Extracción en Fase SólidaRESUMEN
Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate.
Asunto(s)
Escherichia coli/metabolismo , Péptido Natriurético Encefálico/biosíntesis , Péptido Natriurético Encefálico/química , Péptido Natriurético Encefálico/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Humanos , Hidrólisis , Péptido Natriurético Encefálico/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
The pool of peptides composing a protein allows for its distinctive identification in a process named fingerprint (FP) analysis. Here, the FP concept is used to develop a method for the rational preparation of molecularly imprinted polymers (MIPs) for protein recognition. The fingerprint imprinting (FIP) is based on the following: (1) the in silico cleavage of the protein sequence of interest with specific agents; (2) the screening of all the peptide sequences generated against the UniProtKB database in order to allow for the rational selection of distinctive and unique peptides (named as epitopes) of the target protein; (3) the selected epitopes are synthesized and used as templates for the molecular imprinting process. To prove the principle, NT-proBNP, a marker of the risk of cardiovascular events, was chosen as an example. The in silico analysis of the NT-proBNP sequence allowed us to individuate the peptide candidates, which were next used as templates for the preparation of NT-pro-BNP-specific FIPs and tested for their ability to bind the NT-proBNP peptides in complex samples. Results indicated an imprinting factor, IF, of ~10, a binding capacity of 0.5-2 mg/g, and the ability to rebind 40% of the template in a complex sample, composed of the whole digests of NT-proBNP.
Asunto(s)
Impresión Molecular/métodos , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Péptidos/química , Polímeros/química , Secuencia de Aminoácidos , Enfermedades Cardiovasculares/diagnóstico , Humanos , Péptido Natriurético Encefálico/aislamiento & purificación , Péptido Natriurético Encefálico/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Polímeros/metabolismo , Unión ProteicaRESUMEN
The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3fmol/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC-MS.
Asunto(s)
Química Encefálica , Cromatografía Liquida/métodos , Péptido Natriurético Encefálico/química , Péptido Natriurético Encefálico/farmacocinética , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptido Natriurético Encefálico/aislamiento & purificaciónRESUMEN
C-terminal brain (B-type) natriuretic peptide (BNP)-32 is a widely used clinical biomarker for the diagnosis, prognosis, and treatment of heart failure (HF). The 32-aa peptide is synthesized primarily in the atrial and ventricular myocardium and constitutes the mature biologically active form of immature BNP (pro-BNP). There has been mounting evidence that suggests BNP circulates in different structural forms that impact HF diagnosis and in vivo activity. Herein, we have developed and used an immunoaffinity purification assay to isolate endogenous BNP-32 from New York Heart Association class IV patient plasma for subsequent analysis by nano-liquid chromatography (LC) electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS. We have introduced stable isotope-labeled BNP-32 to the assayed plasma to enable quantification of endogenous levels of BNP-32. Unlike the chemically nonspecific point-of-care tests (POCTs) and RIAs used worldwide to quantify BNP-32 from plasma, FT-ICR-MS (unprecedented mass measurement accuracy) coupled with LC (retention time) affords extraordinary molecular specificity, and when combined with the use of internal standards is able to confidently identify and quantify BNP-32. The significance of this work is despite exceedingly high circulating levels of BNP-32 in the New York Heart Association class IV patients as determined by POCTs (>290 fmol/ml) nano-LC-electrospray ionization-FT-ICR-MS data did not reveal any endogenous BNP-32. These results provide molecularly specific evidence for the absence of circulating BNP-32 in advanced-stage HF patients and suggest the existence of altered forms of BNP that are contributing to the POCT values.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Péptido Natriurético Encefálico/sangre , Péptido Natriurético Encefálico/aislamiento & purificación , Cromatografía Liquida , Femenino , Humanos , Marcaje Isotópico , Masculino , Espectrometría de MasasRESUMEN
To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.