Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labels─horseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)─UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.
- MeSH
- imunoanalýza metody MeSH
- imunosorbenty * MeSH
- lidé MeSH
- limita detekce MeSH
- magnetismus MeSH
- nanočástice * MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.
- MeSH
- imunosorbenty MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory * diagnóza MeSH
- nanočástice * chemie MeSH
- oxid křemičitý chemie MeSH
- polyethylenglykoly chemie MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Zvýšené koncentrace exogenního biotinu jsou potenciálním zdrojem interferencí imunoanalytických stanovení, založených na využití reakce streptavidin-biotin. Interference jsou negativní u nekompetitivních (sendvičových) metod a pozitivní u metod kompetitivních. Zvýšení koncentrací biotinu v séru vlivem terapie některých nemocí a vlivem používání řady doplňků stravy a kosmetických přípravků může být extrémně vysoké (300 až 10000násobné). Interference biotinu jsou silně závislé nejen na dávce a sérové koncentraci biotinu, ale i na typu použité metody a měřící platformy. Potenciální interference biotinu se mohou týkat více než 50 analytů včetně steroidních hormonů, kardiálních markerů, léčiv a tumorových markerů. Závažnost problému interference biotinu vedla ke vzniku několika důležitých dokumentů edukačního charakteru a zásadního informačního významu, které jsou v práci zmíněny a komentovány.
Elevated concentrations of exogenous biotin in serum makes interferences in immunoanalytical measurements based on using strepravidin-biotin linkage. Interferences are negative in cases of noncompetitive methods and positive in competitive methods. Elevation of serum biotin causes its use in therapy and very often in cases using as food supplements. Elevation of biotin may be 300-10000 times higher than concentration without addition of exogenous biotin. Interferences of biotin are extremely dependent on method and analytes. Potential interferences are possible in more than 50 analytes measured by immunoanalytical methods, namely of steroid hormones, cardiac markers, tumour markers, some therapeutic drugs and others. Necessity of biotin interference problem lead to creating some important guidance's, warnings and requirements introduced in this work.
- Klíčová slova
- interference,
- MeSH
- biotin * krev MeSH
- chybná diagnóza * MeSH
- imunoanalýza * MeSH
- lidé MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
Herein, we report a novel concept of low-cost flexible platform for fluorescence-based biosensor. The surface of polyethylene naphthalate (PEN) foil was exposed to KrF excimer laser through a photolitographic contact mask. Laser initiated surface modification resulted in micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. High affinity binding protein (albumin-binding domain (ABD) of protein G, Streptococcus G148) recognizing human serum albumin (HSA), genetically fused with streptavidin (SA-ABDwt), was immobilized on the micro-patterned surface through biotin-streptavidin coupling. Fluorescently labelled HSA analyte was detected in several blocking environments, in 1% bovine serum albumin (BSA) and 6% fetal serum albumin (FBS), respectively. We conclude that the presented novel concept enabled us to micropattern functional biosensing layers on the surface of PEN foil in a fast and easy way. It brings all necessary aspects for continuous roll-to-roll fabrication of low-cost optical bioanalytical devices.
- MeSH
- biotin metabolismus MeSH
- fotoelektronová spektroskopie MeSH
- lidé MeSH
- mikrotechnologie metody MeSH
- naftaleny chemie MeSH
- optické jevy * MeSH
- polyethyleny chemie MeSH
- povrchové vlastnosti MeSH
- sérový albumin metabolismus MeSH
- streptavidin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.
- MeSH
- chromatografie kapalinová MeSH
- interakční proteinové domény a motivy genetika MeSH
- lidé MeSH
- mikrofilamentové proteiny genetika izolace a purifikace MeSH
- nádory genetika patologie MeSH
- povrchová plasmonová rezonance * MeSH
- proteiny s doménou LIM genetika izolace a purifikace MeSH
- streptavidin chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Accurate and rapid diagnosis of prosthetic joint infection (PJI) is vital for rational and effective therapeutic management of this condition. Several diagnostic strategies have been developed for discriminating between infected and noninfected cases. However, none of them can reliably diagnose the whole spectrum of clinical presentations of PJI. Here, we report a new method for PJI detection based on magnetically assisted surface enhanced Raman spectroscopy (MA-SERS) using streptavidin-modified magnetic nanoparticles (MNP@Strep) whose surface is functionalized with suitable biotinylated antibodies and then coated with silver nanoparticles by self-assembly. The high efficiency of this approach is demonstrated by the diagnosis of infections caused by two bacterial species commonly associated with PJI, namely, Staphylococcus aureus and Streptococcus pyogenes. The method's performance was verified with model samples of bacterial lysates and with four real-matrix samples of knee joint fluid spiked with live pathogenic bacterial cells. This procedure is operationally simple, versatile, inexpensive, and quick to perform, making it a potentially attractive alternative to established diagnostic techniques based on Koch's culturing or colony counting methods.
Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.
- MeSH
- DNA chemie metabolismus MeSH
- imobilizované proteiny chemie metabolismus MeSH
- magnetismus MeSH
- mikrosféry * MeSH
- mikroskopie elektronová rastrovací MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- streptavidin chemie metabolismus MeSH
- techniky amplifikace nukleových kyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized "homemade" particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.
- MeSH
- diagnostické techniky molekulární metody MeSH
- lidský papilomavirus 16 chemie genetika izolace a purifikace MeSH
- magnetické nanočástice chemie MeSH
- onkogenní proteiny virové chemie genetika MeSH
- polymerázová řetězová reakce metody MeSH
- represorové proteiny chemie genetika MeSH
- streptavidin chemie MeSH
- Publikační typ
- časopisecké články MeSH
For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biotin chemie MeSH
- biotinylace MeSH
- buněčné linie MeSH
- dendrimery chemická syntéza farmakologie MeSH
- epitelové buňky cytologie účinky léků MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- nanočástice chemie ultrastruktura MeSH
- polyaminy chemie MeSH
- polyethylenglykoly chemie MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sérový albumin chemie MeSH
- streptavidin chemie MeSH
- Streptomyces genetika metabolismus MeSH
- systémy cílené aplikace léků * MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
In this study, we investigate the preparation of surface pattern of functional groups on poly(lactide) (PLA) surfaces through the controlled deposition of core-shell self-assemblies based on functionalized PLA-b-PEO amphiphilic block copolymers from selective solvents. Through grafting RGDS peptide onto the functionalized copolymer surface, the presented approach enables to prepare PLA surfaces with random and clustered spatial distribution of adhesive motifs. The proposed topography of the adhesion motif was proved by atomic force microscopy techniques using biotin-tagged RGDS peptide grafted on the surface and streptavidin-modified gold nanospheres which bind the tagged RGDS peptides as a contrast agent. The cell culture study under static and dynamic conditions with MG63 osteosarcoma cell line showed that the clustered distribution of RGDS peptides provided more efficient initial cell attachment and spreading, and resistance to cell detachment under dynamic culture compared to randomly distributed RGDS motif when with the same average RGDS peptide concentration.
- MeSH
- biomimetika MeSH
- buněčná adheze účinky léků MeSH
- kovové nanočástice MeSH
- laktáty chemie MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- nádorové buněčné linie MeSH
- nanostruktury chemie MeSH
- oligopeptidy MeSH
- polyethylenglykoly chemie MeSH
- povrchové vlastnosti MeSH
- streptavidin chemie MeSH
- vazba proteinů MeSH
- zlato MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
