A study on the response of FRET based DNA aptasensors in intracellular environment

Abstract

this paper presents a study of the response of fRet based DnA aptasensors in the intracellular environment. Herein, we extend previous studies of aptasensors functioning in the extracellular environment to detection of antigens in the intracellular environment. An essential step in this research is the use of a novel means of achieving the endocytosis of aptasensors. Specifically, it is demonstrated that functioning aptasensors are successfully endocytosed by functionalizing the aptasensors with endocytosis-inducing DSS peptides. Biomarker proteins are an important indicator of the presence of a specific disease condition in the physiological system. Hence, sensors for biomarker protein detection are being widely studied today. Techniques like Enzyme Linked Immunosorbent Assays (ELISAs) have been traditionally used to determine biomarker proteins like Tumor Necrosis Factor-alpha (TNF-α). TNF-α has been determined to be an important biomarker for infectious conditions like sepsis 1 while Glycated albumin (GA) has been observed to be a more versatile biomarker of diabetes mellitus for all kinds of patients including the ones with blood based disorders 2. Therefore, the objective of this study is to detect these biomarkers in an intracellular environment in order to facilitate early detection of these diseases; this is possible due to the pioneering work on the use of DSS as an endocytosis-inducing peptide. Ghosh et.al have summarized previously published detection techniques for TNF-α and GA and compared the advantages of their FRET based DNA aptasensors with those of the published sensing platforms 1,2. DNA aptamers are short oligonucleotides, which have the capability of binding to another molecule. Aptamer based sensors have been evaluated to have excellent recognition capacity towards various types of target molecules. These antigens include metal ions like lead 3 , mercury 4 , potassium 5-7 as well as biomolecules like thrombin 8 , interferon-γ 9 , ATP 10 , AMP 11 etc. Fluorescence resonance energy transfer (FRET) is a process, which facilitates transfer of energy from a 'donor' nanostructure to an 'acceptor' nanostructure. The efficiency of the FRET process is proportional to 1/ {1 + (d/d o) 6 } where d is the distance between the donor and the acceptor and d o is generally approximately 5 nm 12-14. The 1/{1 + (d/d o) 6 } dependence results from a dipole-dipole interactions between the donor and the acceptor. For a typical case, the FRET effect is relatively strong when d is less than about 5 nm and is relatively weak when d is greater than about 5 nm. In this study, the donor is a semiconductor quantum dot (QD) and the acceptor is a gold (Au) nanoparticle. When the QD and the Au nanoparticle are closer than about 5 nm there is strong transfer of energy from the QD to the Au nanoparticle and there is consequently less energy available in the quantum dot to emit as photons. Accordingly, when the QD and the Au nanoparticle are closer together the light emitted by the QD decreases significantly. The quantum dot ...

Recent Advances in Nanomaterial-Based Aptasensors in Medical Diagnosis and Therapy

“…This system can recognize and imaging prostate cancer cells that express prostate specific membrane antigen (PSMA) protein through delivery of Dox via activation of fluorescence QD. Recently, FRET aptasensors synthesized by conjugating Apt–QDs–AuNPs have been used for the detection of Staphylococcus aureus [ 60 ] and Tumor Necrosis Factor-alpha (TNF-α) [ 61 ]. The authors observed that selective binding occurred with limit of detection of 2 cfu/mL for S. aureus and strong fluorescence of an aptamer–QD conjugated donor was successfully quenched by an AuNPs acceptor in the presence of target molecules.…”

Recent Advances in Aptasensor for Cytokine Detection: A Review

“…Ghosh group developed an aptasensor for TNF-α detection based on FRET in an intracellular environment. The sensor was designed to enable cell-penetrating peptide-induced endocytosis, representing a novel measurement method [71].…”

Aptamer-Conjugated Quantum Dot Optical Biosensors: Strategies and Applications

“…Its broad wavelength absorption spectrum allows PL to be excited in multiple QDs with different colors by a single excitation source. Owing to its broad wavelength absorption spectrum, the QD can utilize distance-dependent energy transfer phenomena, such as fluorescence resonance energy transfer (FRET) and nano surface energy transfer (NSET), through the introduction of other NPs such as AuNP as acceptors [34][35][36]. The high quantum yield of QDs result in high brightness and photochemical stability, making them resistant to chemical and photo degradation [37].…”

“…The second system is based on the materials directly attaching to the aptamer [34,35]. This strategy usually harnesses the distance-dependent energy transfer mechanism.…”

“…are also summarized in Table 1. A QD-NP composite can exhibit the FRET effect in the UV-visible range, where the QD acts as a donor and the NPs act as acceptors that can quench or cause a release of photons [34][35][36]. For example, intracellular tumor necrosis factor-alpha (TNF-α) could be detected using the QD-AuNP sensing material [34].…”