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The harm and impact of the COVID-19 pandemic have highlighted the importance of fast, sensitive, and cost-effective virus detection methods. In this study, we developed a DNA aptamer sensor using nanoparticle-enhanced surface plasmon resonance imaging (SPRi) technology to achieve efficient labeling-free detection of SARS-CoV-2 S protein. We used the same DNA aptamer to modify the surface of the SPRi sensor chip and gold nanoparticles (AuNPs), respectively, for capturing target analytes and amplifying signals, achieving ideal results while greatly reducing costs and simplifying the preparation process. The SPRi sensing method exhibits a good linear relationship (R2 = 0.9926) in the concentration range of 1-20 nM before adding AuNPs to amplify the signal, with a limit of detection (LOD) of 0.32 nM. After amplifying the signal, there is a good linear relationship (R2 = 0.9829) between the concentration range of 25-1000 pM, with a LOD of 5.99 pM. The simulation results also verified the effectiveness of AuNPs in improving SPRi signal response. The SPRi sensor has the advantage of short detection time and can complete the detection within 10 min. In addition, the specificity and repeatability of this method can achieve excellent results. This is the first study to simultaneously capture a viral marker protein and amplify the signal using polyadenylic acid (polyA)-modified DNA aptamers on the SPR platform. This scheme can be used as a fast and inexpensive detection method for diagnosis at the point of care (POC) to combat current and future epidemics caused by the virus.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , COVID-19 , Nanopartículas del Metal , Humanos , Resonancia por Plasmón de Superficie/métodos , Glicoproteína de la Espiga del Coronavirus , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Oro/química , Pandemias , Nanopartículas del Metal/química , COVID-19/diagnóstico , SARS-CoV-2 , ADN , Proteínas ViralesRESUMEN
BACKGROUND: Current solid-phase reversible immobilization (SPRI) beads technology is widely used in molecular biology due to its convenience for DNA manipulation. However, the high performance commercial SPRI beads have no price advantage over our method. Furthermore, the use of commercially available SPRI beads standards does not provide the flexibility required for a number of specific nucleic acid handling scenarios. RESULTS: We report an efficient DNA purification strategy by combining home-made beads-suspension buffer with SPRI beads. The method tests the critical concentrations of polyethylene glycol (PEG) 8000 and beads to maximise recovery. And the composition of the SPRI beads DNA purification system (SDPS) was determined at 20% PEG 8000, 2 M NaCl and 16.3 mM MgCl2, and 1.25 mg/ml beads (1/8th original concentration). Then, we tested the DNA recovery of the SDPS, and the result showed that it was comparable to the control (AMPure XP beads). In the study, we have also developed an adjustment SPRI beads DNA purification system (ASDPS), the volume of ASDPS per reaction is 0.6× reaction volume (beads/samples). The performance of ASDPS is similar to SDPS and the control. But the cost of our methods is only about 1/24th of the control. To further assess its performance, we prepare the DNA-seq libraries to evaluate the yield, library quality, capture efficiency and consistency. We have compared all these results with the performance of the control and confirmed its efficiency. CONCLUSION: We have proposed an alternative DNA purification approach with great flexibility, allowing researchers to manipulate DNA in different conditions. And ultimately, its application will benefit molecular biology research in the future.
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ADN , Análisis Costo-BeneficioRESUMEN
DNA origami synthesis is a well-established technique with wide-ranging applications. In most cases, the synthesized origami must be purified to remove excess materials such as DNA oligos and other functional molecules. While several purification techniques are routinely used, all have limitations, and cannot be integrated with robotic systems. Here the use of solid-phase reversible immobilization (SPRI) beads as a scalable, high-throughput, and automatable method to purify DNA origami is demonstrated. Not only can this method remove unreacted oligos and biomolecules with yields comparable to existing methods while maintaining the high structural integrity of the origami, but it can also be integrated into an automated workflow to purify simultaneously large numbers and quantities of samples. It is envisioned that the SPRI beads purification method will improve the scalability of DNA nanostructures synthesis both for research and commercial applications.
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Neuropilin-1 is transmembrane protein with soluble isoforms. It plays a pivotal role in both physiological and pathological processes. NRP-1 is involved in the immune response, formation of neuronal circuits, angiogenesis, survival and migration of cells. The specific SPRI biosensor for the determination of neuropilin-1 was constructed using mouse monoclonal antibody that captures unbound NRP-1 form body fluids. The biosensor exhibits linearity of the analytical signal between 0.01 and 2.5 ng/mL, average precision value 4.7% and recovery between 97% and 104%. The detection limit is 0.011 ng/mL, and the limit of quantification is 0.038 ng/mL. The biosensor was validated by parallel determination of NRP-1 in serum and saliva samples using the ELISA test, with good agreement of the results.
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Técnicas Biosensibles , Resonancia por Plasmón de Superficie , Animales , Ratones , Resonancia por Plasmón de Superficie/métodos , Neuropilina-1 , Técnicas Biosensibles/métodos , Ensayo de Inmunoadsorción Enzimática , Diagnóstico por ImagenRESUMEN
Follicle-stimulating hormone (FSH) regulates the development, growth, pubertal maturation and reproductive processes of the human body. The determination of serous FSH concentration is significant as an alternative to testicular biopsy in the case of boys suffering from cryptorchidism after orchidopexy, and as a means of determining the menopausal stage in women. The aim of this investigation is to develop a specific array surface plasmon resonance imaging (SPRi) biosensor for the determination of FSH in body liquids such as blood plasma, obtaining sufficient sensitivity to determine FSH at levels characteristic for that hormone in blood plasma, without any signal enhancement. The biosensor consists of a mouse monoclonal anti-FSH antibody attached to the gold surface of a chip via a cysteamine linker. Its linear response range is from 0.08 mIU mL-1 (LOQ) to 20 mIU mL-1, and well covers most of the range of FSH activities found in blood without dilution. The precision of measurement is between 3.2% and 13.1% for model samples, and between 3.7% and 5.6% for spiked plasma samples. Recoveries are in the range from 94% to 108%. The biosensor has good selectivity, and is validated by comparison with ECLE, with good agreement of the results.
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Técnicas Biosensibles , Líquidos Corporales , Masculino , Humanos , Femenino , Animales , Ratones , Hormona Folículo Estimulante , Resonancia por Plasmón de Superficie/métodos , Técnicas Biosensibles/métodos , PlasmaRESUMEN
A new method for the determination of cadherin 12 (CDH12)-an adhesive protein that has a significant impact on the development, growth, and movement of cancer cells-was developed and validated. The method is based on a biosensor using surface plasmon resonance imaging (SPRi) detection. A quartz crystal microbalance was used to analyze the characteristics of the formation of successive layers of the biosensor, from the linker monolayer to the final capture of CDH12 from solution. The association equilibrium constant (KA = 1.66 × 1011 dm3 mol-1) and the dissociation equilibrium constant (KD = 7.52 × 10-12 mol dm-3) of the anti-CDH12 antibody-CDH12 protein complex were determined. The determined analytical parameters, namely the values determining the accuracy, precision, and repeatability of the method, do not exceed the permissible 20% deviations specified by the aforementioned institutions. The proposed method is also selective with respect to possible potential interferents, occurring in up to 100-fold excess concentration relative to the CDH12 concentration. The determined Limit of Quantification (LOQ = 4.92 pg mL-1) indicates the possibility of performing quantitative analysis in human plasma or peritoneal fluid without the need to concentrate the samples; however, particular attention should be paid to their storage conditions, as the analyte does not exhibit high stability. The Passing-Bablok regression model revealed good agreement between the reference method and the SPRi biosensor, with ρSpearman values of 0.961 and 0.925.
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Técnicas Biosensibles , Resonancia por Plasmón de Superficie , Humanos , Resonancia por Plasmón de Superficie/métodos , Líquido Ascítico , Técnicas Biosensibles/métodos , CadherinasRESUMEN
Biomarker measurements are essential for the early diagnosis of complex diseases. However, many current biomarker assays lack sensitivity and multiplexing capacity, work in a narrow detection range and importantly lack real time quality control opportunities, which hampers clinical translation. In this paper, we demonstrate a toolbox to kinetically characterize a biomarker measurement assay using Surface Plasmon Resonance imaging (SPRi) with ample opportunities for real time quality control by exploiting quantitative descriptions of the various biomolecular interactions. We show an accurate prediction of SPRi measurements at both low and high concentrations of various analytes with deviations <5% between actual measurements and predicted measurement. The biphasic binding sites model was accurate for fitting the experimental curves and enables optimal detection of heterophilic antibodies, cross-reactivity, spotting irregularities and/or other confounders. The toolbox can also be used to create a (simulated) calibration curve, enabling calibration-free measurements with good recovery, it allows for easy assay optimizations, and could help bridge the gap to bring new biomarker assays to the clinic.
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Resonancia por Plasmón de Superficie , Resonancia por Plasmón de Superficie/métodos , Calibración , Cinética , Biomarcadores , Control de CalidadRESUMEN
Cortisol is a hormone which plays an essential role in the immune, endocrine, cardiovascular, renal and skeletal systems. Its level increases in response to stress, illness, injury or exhaustion, and it is therefore a significant diagnostic biomarker of stress. An immunosensor for the determination of cortisol by SPRi array was developed. The receptive part of the immunosensor is mouse monoclonal antibody against cortisol, immobilized via cysteamine linker. The optimum pH of the immunosensor is 7.4, and the optimum concentration of the antibody is 50 ng mL-1. The immunosensor is specific for cortisol, and its linear response ranges from 0.20 ng mL-1 (LOQ) to 8 ng mL-1. The precision of the determination was between 3.1% and 3.3%, and the recovery between 99% and 102%. The immunosensor was validated by simultaneous determination of cortisol in serum and saliva samples by a standard method, with good agreement between the results.
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Técnicas Biosensibles , Animales , Ratones , Técnicas Biosensibles/métodos , Hidrocortisona , Inmunoensayo/métodos , Saliva , AnticuerposRESUMEN
A functional cure of hepatitis B virus (HBV) infection or HB antigen loss is rarely achieved by nucleos(t)ide analogs which target viral polymerase. HBx protein is a regulatory protein associated with HBV replication. We thought to identify antiviral compounds targeting HBx protein by analyzing HBx binding activity. Recombinant GST-tagged HBx protein was applied on an FDA-approved drug library chip including 1018 compounds to determine binding affinity by surface plasmon resonance imaging (SPRi) using a PlexArray HT system. GST protein alone was used for control experiments. Candidate compounds were tested for anti-HBV activity as well as cell viability using HepG2.2.15.7 cells and HBV-infected human hepatocytes. Of the 1018 compounds screened, 24 compounds showed binding to HBx protein. Of the top 6 compounds with high affinity to HBx protein, tranilast was found to inhibit HBV replication without affecting cell viability using HepG2.2.15.7 cells. Tranilast also inhibited HBV infection using cultured human hepatocytes. Tranilast reduced HB antigen level dose-dependently. Overall, theSPRi screening assay identified novel drug candidates targeting HBx protein. Tranilast and its related compounds warrant further investigation for the treatment of HBV infection.
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Virus de la Hepatitis B , Hepatitis B , Antivirales/metabolismo , Antivirales/farmacología , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral , ortoaminobenzoatos/farmacologíaRESUMEN
Vascular endothelial growth factor receptor 2 (VEGF-R2) is a marker of angiogenesis and metastasis of cancer. Two biosensors for the determination of VEGF-R2 in plasma have been developed. One of them is based on a pure gold chip, and the other on a silver/gold bimetallic chip; both have the receptor, monoclonal rabbit antibody specific for human VEGF-R2, attached to the chip via a cysteamine linker. The biosensor with the gold chip exhibits linearity of the analytical signal between 0.03 and 2 ng/mL, a precision of 1.4% and recovery between 99% and 102%. The biosensor with the bimetallic chip exhibits linearity between 0.03 and 1 ng/mL, a precision of 2.2% and recovery between 99% and 103%. Both biosensors tolerate a 1:100 excess of VEGF, VEGF-R1 and VEGF-R3. Both biosensors were validated by parallel determination of VEGF-R2 in 27 different plasma samples using the ELISA immunosensor assay, with very good agreement of the results. Thermodynamic parameters of the interaction of VEGF-R2 with the antibody were determined by QCM (Quartz Crystal Microbalance) and SPRi (Surface Plasmon Resonance imaging) measurements.
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Técnicas Biosensibles , Animales , Humanos , Conejos , Técnicas Biosensibles/métodos , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Inmunoensayo , Oro/químicaRESUMEN
We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5γ) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5γ (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo.
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Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Resonancia por Plasmón de Superficie/métodos , ARN Helicasas DEAD-box/metabolismo , Mitocondrias/metabolismo , Proteolisis , ARN Catalítico , Saccharomyces cerevisiae/metabolismoRESUMEN
Human epididymis protein 4 (HE4) is an ovarian cancer marker. Various cut-off values of the marker in blood are recommended, depending on the method used for its determination. An alternative biosensor for HE4 determination in blood plasma has been developed. It consists of rabbit polyclonal antibody against HE4, covalently attached to a gold chip via cysteamine linker. The biosensor is used with the non-fluidic array SPRi technique. The linear range of the analytical signal response was found to be 2-120 pM, and the biosensor can be used for the determination of the HE4 marker in the plasma of both healthy subjects and ovarian cancer patients after suitable dilution with a PBS buffer. Precision (6-10%) and recovery (101.8-103.5%) were found to be acceptable, and the LOD was equal to 2 pM. The biosensor was validated by the parallel determination of a series of plasma samples from ovarian cancer patients using the Elecsys HE4 test and the developed biosensor, with a good agreement of the results (a Pearson coefficient of 0.989). An example of the diagnostic application of the developed biosensor is given-the influence of ovarian tumor resection on the level of HE4 in blood serum.
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Técnicas Biosensibles , Neoplasias Ováricas , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/análisis , Biomarcadores de Tumor/análisis , Femenino , Humanos , Neoplasias Ováricas/diagnóstico , PlasmaRESUMEN
Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.
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Antígeno Carcinoembrionario/sangre , Resonancia por Plasmón de Superficie/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/sangre , Antígeno Ca-125/química , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/diagnóstico , Humanos , Inmunoensayo , Leptina/química , Límite de Detección , Proteínas de la Membrana/química , Inhibidor Tisular de Metaloproteinasa-1/químicaRESUMEN
Based on the hydrogel-AuNP supramolecular sphere (H-Au), a label-free and real-time surface plasmon resonance imaging biosensor has been developed for highly sensitive and specific determination of prostate cancer cell-derived exosomes. After integrating the signal amplification effect of the mass cumulative hydrogel and the LSPR effect of AuNPs with high specific aptamer, the SPRi biosensor for exosome detection exhibited a wide linear range from 1.00 × 105 to 1.00 × 107 particles/mL with a limit of detection of 1.00 × 105 particles/mL. Most importantly, with a strong correlation between the SPRi signal and the t-PSA value measured by the clinical chemiluminescence immunoassay, this biosensor displayed excellent practicability for human serum analysis, which exhibits great potential applications in disease diagnosis and bioanalysis. Prostate cancer has been one of the most threatening diseases in human life and health nowadays. In particular, as cancer metastasizes, it is more likely to cause fracture, paraplegia, and even fatal consequences. However, the predominant t-PSA test needs further improvement for the deficiencies of limited specificity and sensitivity, which is prone to false positive. As one of the noninvasive markers of liquid biopsies, exosome has the potential to be a substitute for t-PSA, which can provide specific and predictive information in disease diagnosis and prognosis. Herein, based on the hydrogel-AuNP supramolecular sphere (H-Au), a label-free and real-time surface plasmon resonance biosensor has been developed for highly sensitive and specific detection of prostate cancer cell-derived exosomes. After integrating the signal amplification effect of mass cumulative hydrogel and LSPR effect of AuNPs with high specific aptamer, this developed SPRi biosensor for exosome detection exhibited a wide linear range from 1.00 × 105 to 1.00 × 107 particles/mL with a limit of detection down to 1.00 × 105 particles/mL. Most importantly, with a strong correlation between the SPRi signal and the t-PSA value measured by the clinical chemiluminescence immunoassay, this biosensor displayed excellent practicability in human serum, which exhibited great potential applications in disease diagnosis and bioanalysis.
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Exosomas/patología , Hidrogeles/química , Nanopartículas del Metal/química , Neoplasias de la Próstata/patología , Antígenos de Superficie/química , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Línea Celular Tumoral , ADN/química , Exosomas/química , Glutamato Carboxipeptidasa II/química , Oro/química , Humanos , Límite de Detección , Masculino , Neoplasias de la Próstata/sangre , Resonancia por Plasmón de SuperficieRESUMEN
Plasma transfusion induces some transfusion related acute lung injury (TRALI) mediated through neutrophil extracellular traps (NETs). We investigated whether extracellular vesicles (EVs) present in plasma or obtained from resting (N-PEVs) or thrombin activated platelets (T-PEVs) can trigger NETs, and whether 75â¯nm-nanofiltration, to partially remove EVs, prohibits NETs formation. EVs size and concentration were determined by conventional biophysical approaches and by an original NanoBioAnalytical (NBA) platform based on EV immunocapture biochip, combining Surface Plasmon Resonance Imaging (SPRi) and Atomic Force Microscopy (AFM) exploration. EVs effective diameter was in the 25-1000â¯nm range, with a majority (≈ 90%)â¯≤â¯100â¯nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70â¯nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.
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Transfusión Sanguínea , Vesículas Extracelulares/metabolismo , Nanotecnología/métodos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Agregación Celular/efectos de los fármacos , Vesículas Extracelulares/efectos de los fármacos , Filtración , Humanos , Nanopartículas/química , Nanoporos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacologíaRESUMEN
Label-free evaluation and monitoring of living cell conditions or functions by means of chemical and/or physical sensors in a real-time manner are increasingly desired in the field of basic research of cells and clinical diagnosis. In order to perform multi-parametric analysis of living cells on a chip, we here developed a surface plasmon resonance (SPR) imaging (SPRI)-impedance sensor that can detect both refractive index (RI) and impedance changes on a sensor chip with comb-shaped electrodes. We then investigated the potential of the sensor for label-free and real-time analysis of living cell reactions in response to stimuli. We cultured rat basophilic leukemia (RBL)-2H3 cells on the sensor chip, which was a glass slide coated with comb-shaped electrodes, and detected activation of RBL-2H3 cells, such as degranulation and morphological changes, in response to a dinitro-phenol-conjugated human serum albumin (DNP-HSA) antigen. Moreover, impedance analysis revealed that the changes of impedance derived from RBL-2H3 cell activation appeared in the range of 1 kHz-1 MHz. Furthermore, we monitored living cell-derived RI and impedance changes simultaneously on a sensor chip using the SPRI-impedance sensor. Thus, we developed a new technique to monitor both impedance and RI derived from living cells by using a comb-shaped electrode sensor chip. This technique may enable us to clarify complex living cell functions which affect the RI and impedance and apply this to medical applications, such as accurate clinical diagnosis of type I allergy.
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Técnicas Biosensibles , Fenómenos Fisiológicos Celulares , Rastreo Celular/métodos , Diagnóstico por Imagen/métodos , Animales , Humanos , Leucemia/diagnóstico , Leucemia/patología , Ratas , Resonancia por Plasmón de SuperficieRESUMEN
An integrated and miniaturized Micro-Gas Chromatography with real-time imaging capability for simultaneous chemical separation and detection was developed. Surface Plasmon Resonance imaging (SPRi) was used as a sensitive and real-time imaging based detector for various gaseous chemical mixtures and good gas chromatographs were obtained. The system integrated a home-made miniaturized molecular sieve packed spiral micro-channel column with the SPRi imaging chip and real-time chemical separation and detection were demonstrated using alkanes. The chemical separation processes were simulated using COMSOL and matched well with experimental results. The system enabled the study of chemical separation processes in real-time by miniaturizing and integrating the Micro-GC separation and detection units. This approach can be expanded to multidimensional GC development.
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Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation.
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Separación Celular , Resonancia por Plasmón de Superficie , Análisis de Matrices Tisulares , Animales , Separación Celular/instrumentación , Separación Celular/métodos , Humanos , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/métodos , Análisis de Matrices Tisulares/instrumentación , Análisis de Matrices Tisulares/métodosRESUMEN
Surface plasmon resonance imaging (SPRi) is a label-free detection method that offers a suitable and reliable platform for the real time monitoring of biomolecular interactions. In the work reported here, SPRi was used to evaluate the affinity and specificity of three different aptamers selected against the Lup an 1 anaphylactic allergen ß-conglutin (ß-conglutin binding aptamers I and II (ß-CBA I and ß-CBA II)), as well as an 11-mer truncated version of ß-CBA I. Thiol modified aptamers were immobilised on a gold substrate through a self-assembling process and the use of different blocking strategies to prevent non-specific binding were evaluated. Dissociation constants of 20, 13 and 1 nM were determined for ß-CBA I, ß-CBA II and the 11-mer truncated aptamer, respectively. The three aptamers were then studied in various different sandwich formats and the ß-CBA I/11-mer and ß-CBA II were observed to bind to different aptatopes on the target protein. Each of the aptamers were then used either as surface immobilised aptamer, or as reporter aptamer, and added with the protein target ß-conglutin in either a sequential of simultaneous manner, and the changes in SPR signal monitored. The preferred approach for formation of a sandwich aptacomplex was with immobilised ß-CBA II, followed by addition of pre-incubated ß-conglutin and 11-mer, whilst addition of the 11-mer following addition of the ß-conglutin, resulted in displacement of the bound target. The ability to provide parallel qualitative and quantitative detection establishes SPRi as a powerful tool for the study of immobilised aptamer-target interactions.
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Aptámeros de Nucleótidos/química , Proteínas de Almacenamiento de Semillas/química , Unión Proteica , Técnica SELEX de Producción de Aptámeros , Resonancia por Plasmón de SuperficieRESUMEN
We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm2. Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.