Label-free protein detection based on the heat-transfer method-a case study with the peanut allergen Ara h 1 and aptamer-based synthetic receptors

© 2015 American Chemical Society. Aptamers are an emerging class of molecules that, because of the development of the systematic evolution of ligands by exponential enrichment (SELEX) process, can recognize virtually every target ranging from ions, to proteins, and even whole cells. Although there are many techniques capable of detecting template molecules with aptamer-based systems with high specificity and selectivity, they lack the possibility of integrating them into a compact and portable biosensor setup. Therefore, we will present the heat-transfer method (HTM) as an interesting alternative because this offers detection in a fast and low-cost manner and has the possibility of performing experiments with a fully integrated device. This concept has been demonstrated for a variety of applications including DNA mutation analysis and screening of cancer cells. To the best our knowledge, this is the first report on HTM-based detection of proteins, in this case specifically with aptamer-type receptors. For proof-of-principle purposes, measurements will be performed with the peanut allergen Ara h 1 and results indicate detection limits in the lower nanomolar regime in buffer liquid. As a first proof-of-application, spiked Ara h 1 solutions will be studied in a food matrix of dissolved peanut butter. Reference experiments with the quartz-crystal microbalance will allow for an estimate of the areal density of aptamer molecules on the sensor-chip surface

Interleukin 6 Accelerates Mortality by Promoting the Progression of the Systemic Lupus Erythematosus-Like Disease of BXSB. Yaa Mice

IL6 is a multifunctional cytokine that drives terminal B cell differentiation and secretion of immunoglobulins. IL6 also cooperates with IL21 to promote differentiation of CD4(+) T follicular helper cells (TFH). Elevated serum levels of IL6 correlate with disease flares in patients with systemic lupus erythematosus (SLE). We previously reported that IL21 produced by T-FH plays a critical role in the development of the SLE-like disease of BXSB. Yaa mice. To examine the possible contributions of IL6 to disease, we compared disease parameters in IL6-deficient and IL6-competent BXSB. Yaa mice. We report that survival of IL6-deficient BXSB. Yaa mice was significantly prolonged in association with significant reductions in a variety of autoimmune manifestations. Moreover, B cells stimulated by co-engagement of TLR7 and B cell receptor (BCR) produced high levels of IL6 that was further augmented by stimulation with Type I interferon (IFN1). Importantly, the frequencies of T-FH and serum levels of IL21 were significantly reduced in IL6-deficient mice. These findings suggest that high-level production of IL6 by B cells induced by integrated signaling from the IFN1 receptor, TLR7 and BCR promotes the differentiation of IL21-secreting T-FH in a signaling sequence that drives the lethal autoimmune disease of BXSB. Yaa mice.Peer reviewe

Impedimetric, diamond-based immmunosensor for the detection of C-reactive protein

The high prevalence of cardiovascular diseases (CVD) demands a reliable and sensitive risk assessment technique. In order to develop a fast and label-free immunosensor for C-reactive protein (CRP), a risk factor for this condition, anti-CRP antibodies were physically adsorbed to the hydrogen (H)-terminated surface of nanocrystalline diamond (NCD). An Enzyme-Linked ImmunoSorbent Assay (ELISA) reference technique showed that this was a suitable substrate for antibody-antigen recognition reactions. Electrochemical Impedance Spectroscopy (EIS) was used to electronically detect CRP recognition. The specificity of the immunosensor was demonstrated by incubation with CRP and plasminogen as reference molecule. A different impedance behavior was observed in real-time after CRP addition as compared to plasminogen addition: the impedance increased only during CRP incubation. Fitting the data showed that this corresponded with a decrease in capacitance of the molecular layer due to its increased thickness by specific CRP recognition. Sensitivity experiments in real-time showed a clear discrimination between 1 μM, 100 nM, and 10 nM of CRP after 10 min at 100 Hz. Since, 10 nM of CRP was still clearly distinguishable from buffer solution, our CRP-directed immunosensor prototype reaches a sensitivity that is within the physiologically relevant concentration range of this biomarker in healthy controls and CVD patients. Moreover, this prototype displayed real-time discriminating power between spiked and unspiked serum, and thus also shows its applicability in this biological matrix. © 2011 Elsevier B.V. All rights reserved.status: publishe

Real-time study of protein adsorption on thin nanocrystalline diamond

The study of protein adsorption on solid surfaces is interesting for theoretical and practical bio-analytical sensing applications. In this work we combine electrochemical impedance spectroscopy, enzyme linked immunosorbent assay, and fluorescence microscopy with thin boron doped nanocrystalline diamond films to address and study the adsorption behavior of globular proteins (antibodies) on hydrophobic and hydrophilic diamond surfaces. A powerful combination of time resolved impedance spectroscopy and data modeling with equivalent circuits allow a detailed insight in the protein behavior at an interface. It is found that hydrogenated diamond is greatly favorable for impedimetric read-out but causes slight conformational loss of the protein structure and therefore also its biological activity. The oxidized surface allows faster adsorption and a high biological activity but results in smaller impedimetric response. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.status: publishe

Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

Natalie Vanden Bon,1 Bart van Grinsven,2 Mohammed Sharif Murib,2 Weng Siang Yeap,2 Ken Haenen,2,3 Ward De Ceuninck,2,3 Patrick Wagner,2,3 Marcel Ameloot,1 Veronique Vermeeren,1 Luc Michiels11Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 2Institute for Materials Research, Hasselt University, Diepenbeek, Belgium; 3IMOMEC, Diepenbeek, BelgiumAbstract: Conventional neonatal diagnosis of phenylketonuria is based on the presence of abnormal levels of phenylalanine in the blood. However, for carrier detection and prenatal diagnosis, direct detection of disease-correlated mutations is needed. To speed up and simplify mutation screening in genes, new technologies are developed. In this study, a heat-transfer method is evaluated as a mutation-detection technology in entire exons of the phenylalanine hydroxylase (PAH) gene. This method is based on the change in heat-transfer resistance (Rth) upon thermal denaturation of dsDNA (double-stranded DNA) on nanocrystalline diamond. First, ssDNA (single-stranded DNA) fragments that span the size range of the PAH exons were successfully immobilized on nanocrystalline diamond. Next, it was studied whether an Rth change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear Rth shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, Rth was shown to detect prevalent single-nucleotide polymorphisms, c.473G>A (R158Q), c.932T>C (p.L311P), and c.1222C>T (R408W), correlated with phenylketonuria, displaying an effect related to the different melting temperatures of homoduplexes and heteroduplexes.Keywords: mutation detection, heat-transfer resistance, melting temperature, nanocrystalline diamond, persistence lengt