Electroanalysis of pM-levels of urokinase plasminogen activator in serum by phosphorothioated RNA aptamer

Abstract

Protein biomarkers of cancer allow a dramatic improvement in cancer diagnostics as compared to the traditional histological characterisation of tumours by enabling a non-invasive analysis of cancer development and treatment. Here, an electrochemical label-free assay for urokinase plasminogen activator (uPA), a universal biomarker of several cancers, has been developed based on the recently selected uPA-specific fluorinated RNA aptamer, tethered to a gold electrode via a phosphorothioated dA tag, and soluble redox indicators. The binding properties of the uPA-aptamer couple and interference from the non-specific adsorption of bovine serum albumin (BSA) were modulated by the electrode surface charge. A nM uPA electroanalysis at positively charged surfaces, complicated by the competitive adsorption of BSA, was tuned to the pM uPA analysis at negative surface charges of the electrode, being improved in the presence of negatively charged BSA. The aptamer affinity for uPA displayed via the binding/dissociation constant relationship correspondingly increased, ca. three orders of magnitude, from 0.441 to 367. Under optimal conditions, the aptasensor allowed 10(-12)-10(-9) M uPA analysis, also in serum, being practically useful for clinical applications. The proposed strategy for optimization of the electrochemical protein sensing is of particular importance for the assessment and optimization of in vivo protein ligand binding by surface-tethered aptamers.

Application of RNA Aptamers as Recognition Layers for the Electrochemical Analysis of C‐Reactive Protein

“…Since the conjugation of redox label to aptamer probe is a laborious and expensive process, the introduction of electroactive indicator seemed to be more favorable. On the basis of literature recommendations as well as our previous experience , methylene blue was chosen for the electroanalysis of the interaction between RNA probe and CRP.…”

“…This indicated a substantial binding between surface tethered RNA aptamer and CRP. Such a pronounced affinity of RNA probe towards target protein has been already reported for RNA aptamer interaction with urokinase plasminogen activator , theophiline and dopamine . Furthermore, it should be noted that the binding at electrode surface was more efficient than in solution, as for such a RNA aptamer – CRP complex K d of 125 nmol L −1 was reported .…”

Hybridization Biosensors Relying on Electrical Properties of Nucleic Acids

“…It might be expected that the linker removal might allow enhancement of the ET rates up to the theoretically predicted values, and the routinely used C 6 alkanethiol linker was replaced by the phosphorothioated adenosine tag, dA 5 *, in which one of the phosphate oxygens is replaced by sulphur (Figure ) . Phosphorothioated residues can be easily and at low cost introduced enzymatically into NA molecules of any length, and DNA binding to gold through the dA x * provides the improved stability of the formed monolayers due to multiple dA x * tag‐gold attachment points making it also useful for advanced biosensor development .…”

Specific Picomolar Detection of a Breast Cancer Biomarker HER‐2/neu Protein in Serum: Electrocatalytically Amplified Electroanalysis by the Aptamer/PEG‐Modified Electrode

“…In most routinely used approaches, an aptamer sequence is tethered to the electrode through the appropriate linker, and the protein binding to the aptamer is monitored by following the variations in electrochemistry of a soluble redox indicator, which electron transfer (ET) reactions may be either accelerated or impeded by protein binding . The most popular redox indicator used hitherto is a ferri‐/ferrocyanide redox couple, which electrochemistry is sensitive both to the protein charge and extent of the interfacial blocking .…”

“…Motivated by this research and our previous work on pM analysis of urokinase plasminogen activator (uPA) at the RNA aptamer‐electrode, with methylene blue (MB) as a soluble redox indicator, we aimed to use the same approach for electrochemical detection of the Human Epidermal growth factor Receptor‐2 (HER‐2/ neu ) protein. HER‐2/ neu is a 185 kDa transmembrane glycoprotein over‐expressed in 25–30 % of breast cancers that are characterized by aggressive growing and spreading of the tumors and, as a rule, have poor clinical prognosis .…”