Nucleic Acids for Ultra-Sensitive Protein Detection

Abstract

Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of “personalized medicine”. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given.

Immuno-PCR in cancer and non-cancer related diseases: a review

“…For many years, the enzyme-linked immunosorbent assay (ELISA) has been used for this purpose. It is a simple, highly sensitive, and easy standardization method; however, ELISA is not suitable for small molecule or trace substance detection (Janssen et al 2013;Chen et al 2014), or some antigens at low concentration (Malou & Raoult 2011). In this way, a new methodology that combines the specificity of the immunological methods, i.e.…”

“…According to Zhang et al (2001), Niemeyer et al (2001), Niemeyer et al (2005), Wacker et al (2007), Adler et al (2008), and Janssen et al (2013), there are different types of immuno-PCR, i.e. original immuno-PCR (streptavidin (STV)-protein A chimeric fusion protein tags the detection antibody with biotinylated DNA), universal immuno-PCR (the signal generating complex is assembled 'in situ' by subsequent incubation steps of biotinylated detection antibody, STV and biotinylated DNA, either using a non-biotinylated primary and a species specific secondary antibody, or a directly biotinylated primary antibody), qiPCR (sequential, modular, and direct qiPCR), immuno-PCR by proximity ligation, competitive immuno-PCR, direct and indirect sandwich immuno-PCR ('in situ'; by capture) (Figure 1), and magnetoimmuno-PCR (similar to the two-sided, sandwich, immunoassay, but with antibody-functionalized magnetosome conjugates; based on the chemically modified magnetosome nanoparticles bearing STV molecules at the magnetosome membrane).…”

“…(F) Immuno-PCR by proximity ligation (sample is incubated with biotinylated antibodies, streptavidin molecule linked to oligonucleotides and linker oligonucleotides (splint); antibodies will bind pairwise to epitopes on target proteins; each streptavidin-oligonuceotides bind to the biotin of each antibody, forming proximity probes; the extremity of the probes are binded by the linker oligunucleotides; when bound to the specific target, it generates an amplicon that can be amplified using different methodologies, i.e. PCR and qPCR) (adapted from Janssen et al 2013 andGreenwood et al 2015).…”

Emerging Technologies to Increase Ligand Binding Assay Sensitivity

“…Although the IPCR technology has been around for quite some time and appears to be a well-established immunoassay technology (28,31,(33)(34)(35), technical improvements and novel IPCR-based applications addressing the assay sensitivity needs in bioanalysis are still emerging. Many new IPCR applications have been reported for biomarkers.…”

Signal Amplification for Highly Sensitive Immunosensing

“…Afterward, many researchers have worked to improve the assay workflow of immuno-PCR by focusing on the key aspects of DNAantibody conjugates, target binding and assay readout. For example, a streptavidin-protein A fusion protein was constructed as a bridge of biotin-DNA complex and antibody based on the specific bind of protein A to the Fc fragment of IgG and streptavidin to the biotin-DNA complex [60]. To overcome this limitation of availability of the fusion proteins and extend the application of immuno-PCR, streptavidin or avidin was used to join both the biotinylated DNA reporter and the biotinylated antibody [61,62].…”