Herein we report the first serum insulin voltammetric immunosensor for diagnosis of type 1 and type 2 diabetic disorders. The sensor is composed of multiwalled carbon nanotube-pyrenebutyric acid frameworks on edge plane pyrolytic graphite electrodes (PGE/MWNT/Py) to which an anti-insulin antibody was covalently attached. The detection of picomolar levels of serum insulin binding to the surface antibody was achieved by monitoring the decrease in voltammetric current signals of a redox probe taken in the electrolyte solution. This method offered a detection limit of 15 pM for free insulin present in serum. This detection limit was further lowered to 5 pM by designing serum insulin conjugates with poly(acrylic acid)-functionalized magnetite nanoparticles (100 nm hydrodynamic diameter) and detecting the binding of MNP-serum insulin conjugate to the surface insulin-antibody on PGE/MWNT/Py electrodes. When tested on real patient serum samples, the sensor accurately measured insulin levels. To our knowledge, this is the first report of a voltammetric immunosensor capable of both diagnosing and distinguishing the type of diabetes based on serum insulin levels in diabetic patients.

An electrochemical mass sensor for clinically relevant detection of insulin in human serum conjugated to magnetic nanoparticles and captured onto antibody immobilized gold coated quartz resonators is reported for the first time.

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New microarray chip strategies that are sensitive and selective and that can measure low levels of important biomarkers directly in a blood sample are significant for improving human health by allowing timely diagnosis of an abnormal condition. Herein, we designed an antibody–aptamer immunoarray chip to demonstrate simultaneous measurement of blood insulin and glycated hemoglobin (HbA1c) levels relevant to diabetic and prediabetic disorders using a surface plasmon microarray with validation by fluorescence imaging. To accomplish both surface plasmon and fluorescence imaging on the same sample, we decorated magnetite nanoparticles with quantum dots for covalent immobilization of aptamers for subsequent capture and isolation of the aptamers specific for insulin and HbA1c markers from 20-times diluted whole blood samples. Direct clinically relevant analysis, along with fluorescent imaging of the two markers, was achieved by this new immunoarray platform. The limit of detection was 4 pM for insulin and 1% for HbA1c. Examination of cross-talk using thrombin and platelet-derived growth factor confirmed that the designed immunoarray was highly selective for insulin and HbA1c. Surface plasmon kinetic analysis provided apparent binding constants of 0.24 (±0.08) nM and 37 (±3) μM, respectively, for the binding of insulin and HbA1c onto their surface immobilized monoclonal antibodies. Thus, quantitative imaging of ultralow levels of blood biomarker levels with binding kinetics is uniquely obtained in the designed immunoarray chip. In conclusion, this report demonstrates considerable significance of the developed magnetite-quantum dot-bioconjugate strategy for clinical diagnostics of whole blood biomarkers with characterization of molecular binding interactions.

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Due to rapidly rising rates of diabetes and prediabetic conditions worldwide and the associated lethal complications, it is imperative to devise new diagnostic tools that reliably and directly measure insulin levels in clinical samples. Herein, we report a simple and sensitive direct imaging of insulin levels in diabetic patient samples using a surface plasmon resonance microarray imager (SPRi). To enhance sensitivity, we utilized magnetic nanoparticles (MNPs) to capture insulin from serum samples either directly or via a capture antibody immobilized on MNPs. The insulin-captured nanoparticles were allowed to bind surface insulin-antibody for detection from pixel intensity increase using a charge coupled device (CCD) built-in with the SPRi. We have compared the analytical figures-of-merit of the SPRi immunoarray on detecting insulin prepared in various percentages of serum solutions. A four parameter logistic model was used to obtain the best fit of microarray responses with insulin concentration and indicated the cooperative binding of insulin–nanoparticle conjugates to surface antibody in both the buffer insulin and the serum insulin conjugates with MNPs. The cooperativity effect is attributed to the greater association of magnetic nanoparticle-bound insulin molecules with increasing concentration of insulin binding to surface antibody. This is the first report of an SPRi immunoarray to accomplish clinical diagnosis of diabetic and prediabetic conditions based on insulin levels with serum matrix effect analysis and comparison between direct and sandwich insulin assay formats.

Simple construction of biocatalytically active films of cytochrome P450 (CYP) bactosomes is quite useful for low-cost, stereoselective, and nicotinamide adenine dinucleotide phosphate hydride-free drug metabolism assays, biosensing, and biocatalytic applications. We report here real-time monitoring of the formation of biocatalytically active films of membrane-bound human CYP 2C9 or 3A4 expressed with CYP reductase (CPR) in Escherichia coli (so-called bactosomes) on a cysteamine self-assembled monolayer of goldinfused quartz crystals. The CYP 2C9+CPR-containing bactosomes exhibited oxygen reduction currents and metabolite yields greater than those of the CYP 3A4+CPR film. The electrocatalytic property correlated with the greater levels of CPR activity and the amount of CYP 2C9 in the CYP 2C9+CPR bactosomes than in the CYP 3A4+CPR bactosomes. The electron mediating role of CPR in the CYP 2C9 bactosomal film (E°′ = −450 mV vs Ag/AgCl) toward electrocatalytic oxygen reduction and hydroxylation of diclofenac was experimentally identified by comparing the film with bactosomes expressed with either CYP 2C9 (E°′ = −310 mV) or CPR (E°′ = −450 mV). The onset of oxygen reduction potentials correlated with the formal potentials of CYP and CYP+CPR films and revealed the electrocatalysis by CYP alone or in association with CPR. Furthermore, an ∼2-fold increase in the level of 4hydroxydiclofenac product formation supported the favorable role of added catalase (hydrogen peroxide scavenger) in preventing damage by reactive oxygen species to the membrane-bound CYP or CYP+CPR bactosomes. The insignificant role of a peroxide shunt pathway for electrocatalysis in the case of the membrane-bound CYP film alone (unlike membrane-free isolated soluble CYP enzymes) and the electron mediation by CPR from the electrode to initiate CYP catalysis in the CYP+CPR bactosomes were discovered in this study. In conclusion, this report describes voltage-driven biocatalysis by bactosomal CYP films with new mechanistic insights into the formal potentials and electrocatalytic pathways of membrane-bound CYP films either alone or in association with CPR in the membrane.