Fig. 1: We start from hydrogenated diamond, which is made to interact with bioreceptors having a primary amine function. The C-H surface functions interact with the amine to create a covalent bond. This reaction is extremely fast (within seconds to minutes). We can then file micro- or even nano-droplets on the diamond to create as many plots as desired.
In a biosensor, a bioreceptor (enzyme, antibody, DNA probe ...) is hung upon an electronic substrate. When the bioreceptor recognizes a target molecule, the substrate must be able to effectively transduce this event into an interpretable signal. In this context, synthetic diamond film substrates look like ideal substrates. We developed a new manufacturing method that takes much better advantage of diamond strengths than before.
An analytical device, such as a biosensor or a biochip, must meet two requirements: that its probes be well-hung and that its signal is clear. Diamond is a very interesting material because it allows both a strong chemical coupling of the bioreceptor, hence a good immobilization, and an efficient electronic transfer, hence a good transduction of the signal (see insets). Manufacturing methods used so far have two drawbacks: either a low density of probes, or a deterioration of the electronic capabilities of the diamond, due to an insulating organic interface layer.
In collaboration with Institut Néel, we have developed an innovative strategy that overcomes these problems of localization and masking. Our new method allows for (bio)functionalizing the diamond in a single step. The bioreceptors are covalently immobilized, the diamond surface remains active, and one can generate a lot of different plots on the sensor. This massive multiplexing of the chemical information immobilized on the diamond substrate was impossible with conventional techniques. The method is illustrated in Fig.1. It has been confirmed by making DNA and antibody biochips.
Among the probes of interest are redox enzymes, which catalyze the oxidation or reduction of their target. We worked with two of them having a Fe-S heme center: cytochrome C (biomolecule present in mitochondria, involved in cellular respiration), and horseradish peroxidase (which catalyses the reduction of hydrogen peroxide in the presence of a carbon molecule). Cytochrome allowed us to demonstrate the transfer of electrons between the working heart of the enzyme (Fe-S) and diamond. As for the peroxidase, it allowed us to build a sensor capable of determining hydrogen peroxide in solution (Fig. 2). These studies prefigure the design of fuel biocells.
This new chemistry for the development of electrochemical biosensors, currently being implemented in collaboration with a DRT/LIST/LCD team, can develop very different systems:
• biocompatible: artificial retinal implants, human-machine interface;
• substrates for cellular networks or neural networks (filing of a neuron on a plot);
• bio-photoconversion of light energy by photosystem II, which is an enzyme complex involved in photosynthesis and oxygen production;
• detection of synaptic activity via glutamate sensor (glutamate is the neurotransmitter), diamond micro-electrodes directly addressing the synapse.
Fig. 2: During the reduction of hydrogen peroxide H2O2 catalyzed by horseradish peroxidase, a direct electron transfer occurs between the diamond electrode and the peroxidase heme group. The electrochemical reaction is converted into electricity.
Rule #1 – immobilize the biological probe in a "final" manner: the probe concentration should not vary over time.
Rule #2 – immobilize without loss of activity: the recognition activity of bio-receptors must be stabilized in time for a long duration and repeated use, such as real-time monitoring for 10 days of the composition of biological media (urine of patients in intensive care likely to have kidney failure, maturation of beer).
Rule #3 – clearly translate the biological recognition signal (antigen-antibody coupling, complementary DNA single strands, enzyme activity...) through the physico-chemical activity of the supporting material.
Diamond: So sensitive!
Diamond is a semiconductor with a large electronic gap, that is to say, it is nearly insulating in its properties. Heavily boron-doped (1 B atom for 1000 C atoms), it becomes an electronic conductor. Doped diamond is used as an electrode and has properties superior to conventional materials (Pt, C, etc ...), especially electrochemical activity in water: the voltage "window" where it is active is 3.5 V, whereas it is 2.5 V for glassy carbon and less than 2 V for Pt. In addition, eddy currents recorded on the diamond are a hundred times lower than those measured with other electrodes: diamond is so sensitive!
Further reading: E. Vanhove et al., Phys. Stat. Solidi (a) 206 (2009) 2063
Maj : 17/02/2014 (947)