Abu Ali Ibn Sina

DNA–bare gold affinity interactions: mechanism and applications in biosensing

The adsorption of DNA onto gold due to affinity interactions is highly desirable for developing low-cost, convenient and sensitive biosensors. To date, DNA–gold adsorption phenomenon has been demonstrated as one of the most promising physical mechanisms for achieving precise control over unmodified gold nanoparticles (AuNPs) aggregation, and DNA monolayer formation on gold surfaces. The adsorption phenomenon is exquisitely controlled by many factors including intermolecular forces, along with DNA composition and sequence. The understanding and manipulation of these factors have allowed broad biosensing applications and notably, sequence-dependent DNA–gold adsorption which may be highly relevant for DNA methylation detection in cancer. Herein, we review the underlying principles governing DNA–gold adsorption as well as recent biosensing strategies based on differential ssDNA/dsDNA–AuNPs adsorption and sequence-dependent DNA–gold adsorption. Finally, we have also contributed insights regarding the future trend of DNA–gold adsorption-based biosensors.

Real time and label free profiling of clinically relevant exosomes

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(−) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14–35% of their bulk population express HER2.

Detection of aberrant protein phosphorylation in cancer using direct gold-protein affinity interactions

Protein phosphorylation is one of the most prominent post-translational mechanisms for protein regulation, which is frequently impaired in cancer. Through the covalent addition of phosphate groups to certain amino-acids, the interactions of former residues with nearby amino-acids are drastically altered, resulting in major changes of protein conformation that impacts its biological function. Herein, we report that these conformational changes can also disturb the proteins ability to interact with and adsorb onto bare gold surfaces. We exploited this feature to develop a simple electrochemical method for detecting the aberrant phosphorylation of EGFR protein in several lung cancer cell lines. This method, which required as low as 10ng/µL (i.e., 50ng) of purified EGFR protein, also enabled monitoring cell sensitivity to tyrosine kinase inhibitors (TKI) - a common drug used for restoring the function of aberrantly phosphorylated proteins in lung cancer. The reported strategy based on direct gold-protein affinity interactions avoids the conventional paradigm of requiring a phospho-specific antibody for detection and could be a potential alternative of widely used mass spectrometry.

Interfacial nano-mixing in a miniaturised platform enables signal enhancement and in situ detection of cancer biomarkers

Interfacial biosensing performs the detection of biomolecules at the bare-metal interface for disease diagnosis by comparing how biological species derived from patients and healthy individuals interact with bare metal surfaces. This technique retrieves clinicopathological information without complex surface functionalisation which is a major limitation of conventional techniques. However, it is still challenging to detect subtle molecular changes by interfacial biosensing, and the detection often requires prolonged sensing times due to the slow diffusion process of the biomolecules towards the sensor surface. Herein, we report on a novel strategy for interfacial biosensing which involves in situ electrochemical detection under the action of an electric field-induced nanoscopic flow at nanometre distance to the sensing surface. This nanomixing significantly increases target adsorption, reduces sensing time, and enables the detection of small molecular changes with enhanced sensitivity. Using a multiplex electrochemical microdevice that enables nanomixing and in situ label-free electrochemical detection, we demonstrate the detection of multiple cancer biomarkers on the same device. We present data for the detection of aberrant phosphorylation in the EGFR protein and hypermethylation in the EN1 gene region. Our method significantly shortens the assay period (from 40 min and 20 min to 3 minutes for protein and DNA, respectively), increases the sensitivity by up to two orders of magnitude, and improves detection specificity.

A SERS microfluidic platform for targeting multiple soluble immune checkpoints

Immune checkpoint blockade therapies are promising next generation immunotherapeutic treatments for cancer. Whilst sequential solid biopsies are an invaluable source of prognostic information, they are not feasible for monitoring therapeutic outcomes over time. Monitoring soluble immune checkpoint markers expression in body fluids could potentially be a better alternative. Current methods (e.g. ELISA) for detecting immune-checkpoint proteins mostly rely on the use of monoclonal antibodies which are expensive and time-consuming to manufacture and isolate. Herein, we report an integrated surface enhanced Raman scattering (SERS)-microfluidics device for the detection of immune checkpoint proteins which involves the use of i) nano yeast single chain variable fragment (scFv) as a promising alternative to monoclonal antibodies providing high stability at relative low-cost and simplicity for production, ii) graphene oxide functionalised surface to reduces the bio functionalization steps, thus avoiding the general paradigm of biotin-streptavidin chemistry and iii) a microfluidic platform enabling alternating current electrohydrodynamics (ac-EHD) induced nanomixing to enhance the target scFv binding and minimize the non-specific interactions. Specific and multiplex detection of immune checkpoint biomarkers is achieved by SERS based spectral encoding. Using this platform, we successfully demonstrated the detection of clinically relevant soluble immune checkpoints PD-1, PD-L1 and LAG-3 from as low as 100 fg/mL of analytes spiked in human serum.