Reversible, oriented and electrochemically addressed immobilisation of 6xhis tagged molecules


addressedThe recent technology of biochips is mainly based on non-specific immobilization of biomolecules (e.g. nanolitre spotting on preactivated surfaces) and optical detection methods (e.g. fluorescence). Such systems, although already available on market, face a serious limitation concerning their further miniaturization down to the submicron scale. Moreover, the concept of the lab-on-chip based on the above described technology brings limitations which will not fulfil the demands for cheap and sensitive instrument with high degree of operational use and portability.

The solution could be found in hybrid technology based on well-introduced nanolithography, 3D thick film technologies, self-assembling techniques and various modes of electrochemical detection. The key task still not satisfactorily solved is the method of locally specific immobilization of biomolecules on the electrode microarray. The use of nanopipeting as well as other recently developed methods (inject printing, laser beam assisted patterning etc.) brings serious technology complications during the production of the enclosed microchamber system since these biomolecules has to be deposited prior to enclosement of the chip. This can result in the lost of activity, orientation and local specificity of the immobilized species.

The concept of electrochemical biochip system brings many advantages and potentials: (i) the electrochemical methods are highly sensitive; (ii) they can be used for detection of enzymatic redox reactions as well as for hybridization of DNA; (iii) the miniaturization of the conductive parts (electrodes) is already well developed and the solution lies in the field of nanolithography; (iv) the electronic circuits for signal and data processing can be miniaturized and can create the part of lab-on-chip instrument itself; (v) there is a plenty of electrochemical methods which can be used on the same electrode; (vi) electrochemical methods are useful not just for detection of the biomolecule presence or its activity on electrode, but also for locally directed immobilization of biomolecule species on it. An original deposition procedure, that uses chronoamperometry (CA), was developed in our laboratory obtaining oriented and reversible immobilisation of HIS6x-tag engineered proteins and their on-chip purification from bacterial crude extracts. This method started with the electrochemical deposition of multilayers of CYS on Au or Pt surfaces and further chemical synthesis of a spacer with a Ni-NTA end.

Recently, we have designed and manufactured the micro-cell which consists of a micro-array of Au electrodes lithographically patterned on a silicon chip covered by a micro-machined glass reservoir provided with vertical connections to capillaries that allow an external pumped continuous flow of the analyte. In this paper, we aim to demonstrate the use of electrochemical continuous flow micro-cell for obtaining the addressed immobilization on one single electrode element of the micro-array. The demonstration was performed by immobilization of AP with an (HIS)6-tag. CYS EDM and chemical synthesis of the spacer were performed on one single Au element of the micro-array. Afterwards the protein was addressed and immobilized on the Ni-NTA spacer group on that specific planar gold working electrode (Au WE), in few minutes and under flow condition. The presence of the AP on the electrode was verified by detecting its activity upon ascorbate-2P.

Continuous flow micro-cell for electrochemical addressing of engineered bio-molecules - J Maly, M Ilie, V Foglietti, E Cianci, A Minotti, L Nardi, A Masci, W.Vastarella and R.Pilloton - Sensors and Actuators B: Chemical (2005) 111, 317-322

 

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