We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.
- Klíčová slova
- DNA array, Erythrocyte lysate, Low-fouling surface chemistry, Polymer brushes, Surface plasmon resonance imaging, microRNA,
- MeSH
- akrylamidy chemie MeSH
- analýza selhání vybavení MeSH
- biokompatibilní potahované materiály chemická syntéza MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- design vybavení MeSH
- frakcionace buněk MeSH
- komplexní směsi analýza MeSH
- mikro RNA analýza chemie genetika MeSH
- polymery chemie MeSH
- povrchová plasmonová rezonance přístrojové vybavení MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- biokompatibilní potahované materiály MeSH
- komplexní směsi MeSH
- mikro RNA MeSH
- poly(carboxybetaine acrylamide) MeSH Prohlížeč
- polymery MeSH
This review focuses on recent advances in the development of functionalizable antifouling coatings and their applications in label-free optical biosensors. Approaches to the development of antifouling coatings, ranging from self-assembled monolayers and PEG derivatives to ultra-low-fouling polymer brushes, are reviewed. Methods of preparation and characterization of antifouling coatings and the functionalization of antifouling coatings with bioreceptors are reviewed, and the effect of functionalization on the fouling properties of biofunctional coating is discussed. Special attention is given to biofunctional coatings for label-free bioanalysis of blood plasma and serum for medical diagnostics.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- bioznečištění * MeSH
- krevní plazma chemie MeSH
- polymery MeSH
- sérum chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- polymery MeSH
The non-specific binding of non-target species to functionalized surfaces of biosensors continues to be challenge for biosensing in real-world media. Three different low-fouling and functionalizable surface platforms were employed to study the effect of functionalization on fouling resistance from several types of undiluted media including blood plasma and food media. The surface platforms investigated in this work included two polymer brushes: hydroxy-functional poly(2-hydroxyethyl methacrylate) (pHEMA) and carboxy-functional poly(carboxybetaine acrylamide) (pCBAA), and a standard OEG-based carboxy-functional alkanethiolate self-assembled monolayer (AT-SAM). The wet and dry polymer brushes were analyzed by AFM, ellipsometry, FT-IRRAS, and surface plasmon resonance (SPR). The surfaces were functionalized by the covalent attachment of antibodies, streptavidin, and oligonucleotides and the binding and biorecognition characteristics of the coatings were compared. We found that functionalization did not substantially affect the ultra-low fouling properties of pCBAA (plasma fouling of ~20 ng/cm(2)), a finding in contrast with pHEMA that completely lost its resistance to fouling after the activation of hydroxyl groups. Blocking a functionalized AT-SAM covalently with BSA decreased fouling down to the level comparable to unblocked pCBAA. However, the biorecognition capability of blocked functionalized AT-SAM was poor in comparison with functionalized pCBAA. Limits of detection of Escherichia coli O157:H7 in undiluted milk were determined to be 6×10(4), 8×10(5), and 6×10(5) cells/ml for pCBAA, pHEMA, and AT-SAM-blocked, respectively. Effect of analyte size on biorecognition activity of functionalized coatings was investigated and it was shown that the best performance in terms of overall fouling resistance and biorecognition capability is provided by pCBAA.
- Klíčová slova
- Functionalization, Low-fouling coatings, Polymer brushes, Surface chemistry, Surface plasmon resonance sensor,
- MeSH
- adsorpce MeSH
- akrylamidy chemie MeSH
- Escherichia coli izolace a purifikace MeSH
- limita detekce MeSH
- mléko mikrobiologie MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- polymery chemie MeSH
- povrchová plasmonová rezonance metody MeSH
- povrchové vlastnosti MeSH
- sulfhydrylové sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- akrylamidy MeSH
- poly(carboxybetaine acrylamide) MeSH Prohlížeč
- polyhydroxyethylmethakrylát MeSH
- polymery MeSH
- sulfhydrylové sloučeniny MeSH
There is a demand for efficient tools for the monitoring of RNase H activity. We report on a new assay which allows for simultaneous (1) real-time monitoring of RNase H activity and (2) detection of cleavage reaction products. The dual assay is implemented using a multichannel surface plasmon resonance (SPR) biosensor with two independently functionalized sensing areas in a single fluidic path. In the first sensing area the RNA cleavage by RNase H is monitored, while the products of the cleavage reaction are captured in the second sensing area with specific DNA probes. The assay was optimized with respect to AON concentration and temperature. A significant improvement was obtained with special chimeric probes, which contain RNA substrate for RNase H and a longer deoxyribonucleotide tail, which enhances the SPR signal. It has been shown that RNase H stabilizes the RNA:DNA hybrid duplex before the cleavage. The potential of the assay is demonstrated in the study in which the ability of natural and modified oligonucleotides to activate RNase H is examined.
- MeSH
- DNA sondy MeSH
- heteroduplexy nukleové kyseliny MeSH
- povrchová plasmonová rezonance přístrojové vybavení metody MeSH
- proteiny z Escherichia coli analýza metabolismus MeSH
- ribonukleasa H analýza metabolismus MeSH
- RNA sondy MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA sondy MeSH
- heteroduplexy nukleové kyseliny MeSH
- proteiny z Escherichia coli MeSH
- ribonukleasa H MeSH
- RNA sondy MeSH
Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg2+ in buffer led to significantly higher hybridization than even 1 M Na+). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.
- MeSH
- chybné párování bází MeSH
- DNA sondy chemie MeSH
- hořčík chemie MeSH
- hybridizace nukleových kyselin * MeSH
- kationty dvojmocné chemie MeSH
- kationty jednomocné chemie MeSH
- povrchová plasmonová rezonance * MeSH
- sodík chemie MeSH
- spektrofotometrie ultrafialová MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA sondy MeSH
- hořčík MeSH
- kationty dvojmocné MeSH
- kationty jednomocné MeSH
- sodík MeSH
Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats-IN complexes with the host DNA.
- MeSH
- DNA primery MeSH
- HIV-integrasa chemie MeSH
- povrchová plasmonová rezonance metody MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- HIV-integrasa MeSH
- p31 integrase protein, Human immunodeficiency virus 1 MeSH Prohlížeč
Surface plasmon resonance (SPR) biosensors belong to label-free optical biosensing technologies. The SPR method is based on optical measurement of refractive index changes associated with the binding of analyte molecules in a sample to biorecognize molecules immobilized on the SPR sensor. Since late 1990's, SPR biosensors have become the main tool for the study of biomolecular interactions both in life science and pharmaceutical research. In addition, they have been increasingly applied in the detection of chemical and biological substances in important areas such as medical diagnostics, environmental monitoring, food safety and security. This chapter reviews the main principles of SPR biosensor technology and discusses applications of this technology for rapid, sensitive and specific detection of chemical and biological analytes.
- MeSH
- analýza selhání vybavení MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- biotest přístrojové vybavení metody MeSH
- design vybavení MeSH
- imunoanalýza přístrojové vybavení metody MeSH
- povrchová plasmonová rezonance přístrojové vybavení metody MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This paper describes the direct label-free detection of antibodies against the Epstein-Barr virus (anti-EBNA) using a surface plasmon resonance (SPR) biosensor. The antibody detection was performed using the immunoreaction between anti-EBNA and a respective synthetic peptide (EBNA-1), which was conjugated with bovine serum albumin (BSA-EBNA) and immobilized on the sensor surface. Three immobilization chemistries for the attachment of BSA-EBNA were investigated to optimize ligand density and minimize loss of EBNA-1 immunoreactivity. The developed SPR biosensor functionalized with the optimal immobilization method was calibrated and characterized in terms of detection limit, reproducibility, regenerability and storability. It was demonstrated that the sensor is capable of detecting concentrations of anti-EBNA as low as 0.2 ng/ml (approximately 1 pM) both in buffer and 1% human serum and can be stored and regenerated for repeated use.
- MeSH
- analýza selhání vybavení MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- design vybavení MeSH
- imunoanalýza přístrojové vybavení metody MeSH
- protilátky analýza imunologie MeSH
- virus Epsteinův-Barrové - jaderné antigeny analýza imunologie MeSH
- virus Epsteinův-Barrové imunologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- EBV-encoded nuclear antigen 1 MeSH Prohlížeč
- protilátky MeSH
- virus Epsteinův-Barrové - jaderné antigeny MeSH
We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- hybridizace nukleových kyselin metody MeSH
- nanotechnologie přístrojové vybavení metody MeSH
- oligonukleotidové sondy chemie genetika MeSH
- povrchová plasmonová rezonance metody MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- oligonukleotidové sondy MeSH
Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.
