Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.
Pregnancy associated plasma protein A2 (PAPP-A2) is a metalloproteinase that plays multiple roles in fetal development and post-natal growth. Here we present a novel surface plasmon resonance (SPR) biosensor for the rapid and quantitative detection of PAPP-A2 in blood samples. This biosensor uses a single surface referencing approach and a sandwich assay with functionalized gold nanoparticles for signal enhancement. We demonstrate that this SPR biosensor enables the detection of PAPP-A2 in 30 % blood plasma at levels as low as 3.6 ng/mL. We also characterize the performance of the biosensor and evaluate its cross-reactivity to a PAPP-A analogue. Finally, we utilize this SPR biosensor for the detection of PAPP-A2 in blood serum from two groups of subjects: pregnant women and healthy non-pregnant women and men. Graphical Abstract Temporal sensor response corresponding to respective steps of the assay for detection of PAPP-A2 in buffer.
The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (k a 2.0 × 10 5 M 1 s −1 , k d 5.8 × 10 4 s −1 , and K D 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10-cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.
Aiming at developing mechanism-based amino acid (18)F-PET tracers for tumor imaging, we synthesized two (18)F-labeled analogues of 5-hydroxy-l-[β-(11)C]tryptophan ([(11)C]5HTP) whose excellent in vivo performance in neuroendocrine tumors is mainly attributed to its decarboxylation by aromatic amino acid decarboxylase (AADC), an enzyme overexpressed in these malignancies. Reference compounds and precursors were synthesized following multistep synthetic approaches. Radiosynthesis of tracers was accomplished in good radiochemical yields (15-39%), high specific activities (45-95 GBq/μmol), and excellent radiochemical purities. In vitro cell uptake was sodium-independent and was inhibited ≥95% by 2-amino-2-norbornanecarboxylic acid (BCH) and ∼30% by arginine. PET imaging in mice revealed distinctly high tumor/background ratios for both tracers, outperforming the well-established O-(2-[(18)F]fluoroethyl)tyrosine ([(18)F]FET) tracer in a head-to-head comparison. Biological evaluation revealed that the in vivo performance is most probably independent of any interaction with AADC. Nevertheless, the excellent tumor visualization qualifies the new tracers as interesting probes for tumor imaging worthy for further investigation.
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