The 3rd generation biosensor technology

 Biosensors make use of enzymes that specifically detect a substance in a sample and convert it into a measurable signal. For our sensors, we apply a class of engineered enzymes, which are capable of direct electron transfer. This feature allows a 3rd generation glucose sensing mechanism, which is independent of mediators or oxygen. Electrons get directly transfered to a conductive surface such as carbon without further modification needed.

In first-generation biosensors, the electrons are transferred to molecular oxygen and the resulting decrease in the oxygen concentration and/or the produced hydrogen peroxide is measured.

Second-generation biosensors use artificial, partially toxic mediators or nanomaterials to transport the electrons to the electrode.

In third-generation biosensors, the electrons are transferred directly from the enzyme to the electrode without any intermediate stages or use of nanoparticles.

Performance of a Glucose sensor

The special 3rd generation feature makes the technology interesting for the continuous measurement of glucose, such as performed in subcutaneous sensors for diabetes measurement. See below the performance of a sensor setup using common screen printing technology, which is independent of oxygen. The low working potential of -100 mV vs. Ag|AgCl  renders the sensor insensitive towards interfering substances like acetaminophen or ascorbic acid, while other non-electroactive substances  have also been tested negative.

Screen-printed sensor setup

Functional scheme of the sensor set-up consisting of 3 electrodes: A 0.5 mm² carbon working electrode (WE) bearing an optimised enzyme and a 2 mm² carbon counter electrode (CE) operated at -0.1 V vs. Ag|AgCl reference electrode (RE) using an amperometric technique. Electrons gained during glucose conversion are directly transferred to the electrode surface, resulting in analytical currents.

Oxygen independency

Calibration in presence and absence of oxygen: Each calibration represents three independent sensors measured in the presence and absence of oxygen. The red curve describes the current response of sensors submersed in N2-purged PBS buffer, while the black values where recorded without purging. The experimental data show the independency of the sensing mechanism of oxygen.

Interference Studies

Each substance was measured in the presence of 5 mM (90 mg dL-1) glucose at concentrations recommended by the CLSI POCT05-A guideline. The boxplot represents data of 5 repetitions. None of the tested substances altered the sensor readout above 10%. Note, no selective membrane was applied.

Performance

The sensor performance data were evaluated and summarised below. The concentration range with R² values for linear fit above 0.998 is defined as linear range; three-fold background defines the detection limit, one fold background the sensor resolution. Sensitivity is given by the slope of regression normalised by the geometric surface. No algorithms for signal smoothing were used.

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