Graphene paper has diverse applications in printed circuit board electronics, bioassays, 3D cell culture, and biosensing. Although development of nanometal-graphene hybrid composites is commonplace in the sensing literature, to date there are only a few examples of nanometal-decorated graphene paper for use in biosensing. In this manuscript, we demonstrate the synthesis and application of Pt nano cauliflower-functionalized graphene paper for use in electrochemical biosensing of small molecules (glucose, acetone, methanol) or detection of pathogenic bacteria (Escherichia coli O157:H7). Raman spectroscopy, scanning electron microscopy and energy dispersive spectroscopy were used to show that graphene oxide deposited on nanocellulose crystals was partially reduced by both thermal and chemical treatment. Fractal platinum nanostructures were formed on the reduced graphene oxide paper, producing a conductive paper with an extremely high electroactive surface area, confirmed by cyclic voltammetry and electrochemical impedance spectroscopy. To show the broad applicability of the material, the platinum surface was functionalized with three different biomaterials: 1) glucose oxidase (via chitosan encapsulation); 2) a DNA aptamer (via covalent linking), or 3) a chemosensory protein (via his linking). We demonstrate the application of this device for point of care biosensing. The detection limit for both glucose (0.08 ± 0.02 μM) and E. coli O157:H7 (1.3 ± 0.1 CFU mL-1) were competitive with, or superior to, previously reported devices in the biosensing literature. The response time (6 sec for glucose and 10 min for E. coli) were also similar to silicon biochip and commercial electrode sensors. The results demonstrate that the nanocellulose-graphene-nanoplatinum material is an excellent paper-based platform for development of electrochemical biosensors targeting small molecules or whole cells for use in point of care biosensing.
Here we demonstrate a novel approach for fabricating point of care (POC) wearable electrochemical biosensors based on 3D patterning of bionanocomposite networks. To create Bio-Inspired Patterned network (BIPS) electrodes, we first generate fractal network in silico models that optimize transport of network fluxes according to an energy function. Network patterns are then inkjet printed onto flexible substrate using conductive graphene ink. We then deposit fractal nanometal structures onto the graphene to create a 3D nanocomposite network. Finally, we biofunctionalize the surface with biorecognition agents using covalent bonding. In this paper, BIPS are used to develop high efficiency, low cost biosensors for measuring glucose as a proof of concept. Our results on the fundamental performance of BIPS sensors show that the biomimetic nanostructures significantly enhance biosensor sensitivity, accuracy, response time, limit of detection, and hysteresis compared to conventional POC non fractal electrodes (serpentine, interdigitated, and screen printed electrodes). BIPs, in particular Apollonian patterned BIPS, represent a new generation of POC biosensors based on nanoscale and microscale fractal networks that significantly improve electrical connectivity, leading to enhanced sensor performance.
Listeria monocytogenes is one of the most common causes of food illness deaths worldwide, with multiple outbreaks in the United States alone. Current methods to detect foodborne pathogens are laborious and can take several hours to days to produce results. Thus, faster techniques are needed to detect bacteria within the same reliability level as traditional techniques. This study reports on a rapid, accurate, and sensitive aptamer biosensor device for Listeria spp. detection based on platinum interdigitated array microelectrodes (Pt-IDEs). Pt-IDEs with different geometric electrode gaps were fabricated by lithographic techniques and characterized by cyclic voltammetric (CV), electrochemical impedance spectroscopy (EIS), and potential amperometry (DCPA) measurements of reversible redox species. Based on these results, 50 μm Pt-IDE was chosen to further functionalize with a Listeria monocytogenes DNA aptamer selective to the cell surface protein internalin A, via metal-thiol self-assembly at the 5' end of the 47-mer's. EIS analysis was used to detect Listeria spp. without the need for label amplification and pre-concentration steps. The optimized aptamer concentration of 800 nM was selected to capture the bacteria through internalin A binding and the aptamer hairpin structure near the 3' end. The aptasensor was capable of detecting a wide range of bacteria concentration from 10 to 106 CFU/mL at lower detection limit of 5.39 ± 0.21 CFU/mL with sensitivity of 268.1 ± 25.40 (Ohms/log [CFU/mL]) in 17 min. The aptamer based biosensor offers a portable, rapid and sensitive alternative for food safety applications with one of the lowest detection limits reported to date.
Foodborne outbreaks caused by Listeria monocytogenes continue to raise major public health concerns worldwide. In the United States alone, the centers for disease control and prevention have confirmed the occurrence of 183 cases of listeriosis with 39 fatalities within the last 3 years. Standard methods for the detection of pathogenic strains require up to 7 days to yield results, thus faster techniques with the same level of reliability for bacteria detection are desirable. This study reports on the development of a rapid, accurate, and sensitive electrochemical biosensor for rapid testing of Listeria spp. based on the selective binding of InlA aptamers to internalins in the cell membrane of the target bacteria. Hybrid nanomaterial platforms based on reduced graphene oxide and nanoplatinum were deposited onto Pt/Ir electrodes for enhancing electrochemical transduction during the recognition events. InlA aptamers were immobilized onto the nanomaterial platforms via metal-thiol adsorption. Aptamer loading onto different platform nanostructures was investigated through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The detection mechanism was evaluated by recording the electrochemical response to several bacterial dilutions in PBS buffer using the non-pathogenic species Listeria innocua. These preliminary results show that the aptasensor can be tuned for detection of Listeria concentrations as low as 100 CFU/ml in less than 3 hours (including incubation time and data analysis). The developed aptasensor opens a promising direction for rapid testing of Listeria monocytogenes in food products.
In this study, we have designed an electrochemical biosensor for real-time detection of specific biomarkers of bacterial
metabolism related to meat spoilage (hypoxanthine and xanthine). The selective biosensor was developed by assembling
a ‘sandwich’ of nanomaterials and enzymes on a platinum-iridium electrode (1.6 mm tip diameter). The materials
deposited on the sensor tip include amorphous platinum nanoclusters (i.e. Pt black), reduced graphene oxide, nanoceria,
and xanthine oxidase. Xanthine oxidase was encapsulated in laponite hydrogel and used for the biorecognition of
hypoxanthine and xanthine (two molecules involved in the rotting of meat by spoilage microorganisms). The developed
biosensor demonstrated good electrochemical performance toward xanthine with sensitivity of 2.14 ± 1.48 μA/mM,
response time of 5.2 ± 1.5 sec, lower detection limit of 150 ± 39 nM, and retained at least 88% of its activity after 7 days of continuous use.
Advances in biocompatible materials and electrocatalytic nanomaterials have extended and enhanced the field of biosensors. Immobilization of biorecognition elements on nanomaterial platforms is an efficient technique for developing high fidelity biosensors. Single layer (i.e., Langmuir–Blodgett) protein films are efficient, but disadvantages of this approach include high cost, mass transfer limitations, and Vromer competition for surface binding sites. There is a need for simple, user friendly protein-nanomaterial sensing membranes that can be developed in laboratories or classrooms (i.e., outside of the clean room). In this research, we develop high fidelity nanomaterial platforms for developing electrochemical biosensors using sustainable biomaterials and user-friendly deposition techniques. Catalytic nanomaterial platforms are developed using a combination of self assembled monolayer chemistry and electrodeposition. High performance biomaterials (e.g., nanolignin) are recovered from paper pulp waste and combined with proteins and nanomaterials to form active sensor membranes. These methods are being used to develop electrochemical biosensors for studying physiological transport in biomedical, agricultural, and environmental applications.
Non-invasive tools that allow real-time quantification of molecules relevant to metabolism, homeostasis, and cell signaling in cells and tissue are of great importance for studying physiology. Several microsensor technologies have been developed to monitor concentration of molecules such as ions, oxygen, electroactive molecules (e.g., nitric oxide, hydrogen peroxide), and biomolecules (e.g., sugars, hormones). The major challenges for microsensors are overcoming relatively low sensitivity and low signal-to-noise ratio. Modern approaches for enhancing microsensor performance focus on the incorporation of catalytic nanomaterials to increase sensitivity, reduce response time, and increase operating range. To improve signal-to-noise ratio, a non-invasive microsensor modality called self-referencing (SR) is being applied. The SR technique allows measurement of temporal and spatial transport dynamics at the cell, tissue, organ, and organismal level.