Biocompatible hydrogel membranes for the protection of RNA aptamer-based electrochemical sensors

Abstract

Electrochemical-aptamer based (E-AB) sensors represent a universal specific, selective, and sensitive sensing platform for the detection of small molecule targets. Their specific detection abilities are afforded by oligonucleotide (RNA or DNA) aptamers employed as electrode-bound biorecognition elements. Sensor signaling is predicated on bindinginduced changes in conformation and/or flexibility of the aptamer that is readily measurable electrochemically. While sensors fabricated using DNA aptamers can achieve specific and selective detection even in unadulterated sample matrices, such as blood serum, RNA-based sensors fail when challenged in the same sample matrix without significant sample pretreatment. This failure is at least partially a result of enzymatic degradation of the RNA sensing element. This degradation destroys the sensing aptamer inhibiting the quantitative measurement of the target analyte and thus limits the application of E-AB sensors constructed with RNA aptamer. To circumvent this, we demonstrate that a biocompatible hydrogel membrane protects the RNA aptamer sensor surface from enzymatic degradation for at least 3 hours -a remarkable improvement over the rapid (~minutes) degradation of unprotected sensors. To demonstrate this, we characterize the response of sensors fabricated with representative DNA and RNA aptamers directed against the aminoglycoside antibiotic, tobramycin in blood serum both protected and unprotected by a polyacrylamide membrane. Furthermore, we find encapsulation of the sensor surface with the hydrogel does not significantly impede the detection ability of aptamer-based sensors. This hydrogel-aptamer interface will thus likely prove useful for the long-term monitoring of therapeutics in complex biological media.

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA

“…RIs are ~50 kDa proteins that stoichiometrically bind (1:1) to RNases inhibiting nucleolytic activity. 10,24 The observation of protected sensor elements in RI treated serum is consistent with previously published reports 3,12,13,15,25 and is a clear indication that nuclease activity is the dominant cause of sensor failure.…”

“…We previously reported the protection of RNA aptamer sensor surfaces with a polyacrylamide film, however, even with these films, we observed a reduction of ~30% current before stabilization. 15 Here we find that the collagen-based membrane provides superior protection compared to polyacrylamide. However, if we employ the collagen I coated sensors incubated with serum at a pH ~ 10, a pH at which the RI should be inactivate, we observed a significant signal decrease (~30 %) over the time course of 6 hours, similar to the report by Schoukroun-Barnes et al using a polyacrylamide membrane 15 (Figure 6).…”

“…More recently, we demonstrated the usefulness of a polyacrylamide hydrogel membrane to passively protect an aminoglycoside-specific aptamer from nuclease activity in untreated serum. 15 This method demonstrated an initial 30% signal electrochemical signal before stabilizing with the copolymerization of acrylamide and bisacrylamide and only provided protection for a short-time period.…”

Biosensors to Monitor Cell Activity in 3D Hydrogel-Based Tissue Models

“…Moreover, it was demonstrated that the direct contact of aptamer-based electrochemical biosensors with biological complex culture media may affect sensors performance and stability due to the serum protein-dependent degradation of the sensing oligonucleotides; this, in turn, leads to the need for sample pretreatments or more complex chemical modification of the aptamers [239,[266][267][268][269].…”

Detecção de alterações de miRNAs relacionados ao câncer colorretal utilizando sensores de carga e massa

“…Para o estudo mostrado em [39] houve um processo de otimização onde foram testadas diferentes configurações para uma amostra de RNA de 26 bases nitrogenadas, imobilizadas na superfície de um eletrodo de ouro com a finalidade de detectar a ligação de tobramicina, incluindo a variação das frequências de aquisição testadas, e a variação das diferentes formas conformacionais da molécula utilizada como alvo. Para o biossensor otimizado foi encontrada uma faixa dinâmica de 220 nM ~ 42 μM e um limite de detecção de 220nM.…”