Patent Application
20210332489
The present invention relates to apparatus, methods, and systems of making and utilizing electrical and electronic circuits and components and, in particular, in the context of graphene-based electronics and, further, in the context of laser induced graphene (LIG) electrodes that can be used for various applications by effective functionalization of portions of the LIG electrodes. Examples of applications include but are not necessarily limited to electrochemical sensing, catalysis, immunosensors, electrochemical ion sensing, pesticide monitoring, and water splitting.
Graphene-based electronics offer great promise for a wide variety of applications including supercapacitors and batteries, graphene tattoo sensors, and other electronics [1-13]. Challenges with realizing graphene-based electronics lie in relatively complex fabrication procedures, which have generally included chemical vapor deposition (CVD) and/or complex substrate-transfer techniques [14]. However, alternative scalable manufacturing protocols for graphene-based electrical circuits are beginning to emerge, including solution-phase printing (inkjet printing [15, 16], screen printing [17], dispenser printing [18]) and direct laser scribing among others [9, 10]. A promising alternative to printed graphene circuits is laser-induced graphene, which is a one-step lithography-free process upon which a laser converts sp3-hybridized carbon found in substrates, such as polyimide, into sp2-hybridized carbon: the carbon allotrope found in graphene [10, 19, 20]. Laser induced graphene has been used to prevent microbial fouling and in antimicrobial applications [21]. It has also been used for biosensing applications based on ion selective membrane and enzymatic and aptamer binding reactions [4, 14, 22]. However, a laser-induced graphene immunosensor has not been demonstrated.
Biosensors based on impedimetric detection have been designed for a number of pathogens, including Salmonella Typhimurium [23-26], Escherichia coli O157:H7 [27], Listeria monocytogenes [28], and E. coli O111[29], to name a few. The main challenge in the field of biosensing for monitoring pathogens is not response time, but rather the poor detection limit (approximately 102-103 CFU mL−1). To resolve this, most groups have integrated a pre-enrichment step. Although this indeed improves detection limit when only considering signal acquisition, in applied settings this approach trivializes the need for the rapid sensor (the incubation step can take as long as a rtPCR assay). This invention resolves this problem by optimizing the bacteria capture efficiency by developing high surface LIG-based sensors.
This work demonstrates the fabrication of a highly porous, high resolution, thin film (film thickness of ˜25 nm) laser-induced graphene on a polyimide sheet and its application to create in-field electrochemical immunosensor detection of foodborne pathogen, Salmonella enterica. Previous studies have demonstrated highly sensitive and label-free sensors, for example those based on electrodes made of gold nanoparticles[36] and double-walled electrode made of carbon nanotubes[37], but all required time greater than 22 minutes (pre-enrichment step) to perform the test. These steps add complexity to the assay and render the technique difficult for point-of-use applications. Furthermore, the published biosensors that display perfounance similar to this invention use expensive or complex fabrication techniques including chemical vapor deposition or precious metal [24, 25, 38], significantly affecting the device's cost and consequently commercialization.
Both the use of metallic particles or need of pre-treatment of the sample (pre-labeling or pre-enrichment) increase the cost of the biosensor, as compared to the LIG immunosensor which can be used as a one-time, disposable biosensor. In this invention, we demonstrate that this graphene electrode fabrication technique using laser inducing process is capable of sensing Salmonella enterica at low concentration, 13±7 CFU mL−1 in complex media, chicken broth with a response time of around 20 minutes over a broad range of bacteria concentration, from 101 to 105 CFU mL−1 without the need to pre-label or pre-enrichment the sample nor the need to immobilize metallic nanoparticles onto the graphene surface to increase its reactive surface area.
Similar and additional issues have been identified regarding the state of the art of electrode-based technologies, including electrochemical ion sensing, pesticide monitoring, and water-splitting.
This invention introduces a laser-induced graphene electrodes that can be adapted for various useful purposes. A laser is controlled to efficiently and effectively create LIG patterns from a substrate of carbon precursor. The controlled operation of a laser generates LIG patterns which have characteristics and properties that are shown to produce advantageous results as LIG electrodes in a variety of effective ways. The combination of the controlled LIG generation and specific functionalization of at least part of the generated LIG pattern have been demonstrated effective for different applications, inter alia, electrochemical sensing, catalysis, immunosensing, ion sensing, pesticide monitoring, and water splitting.
In one example, the combination is used in biochemical sensing including immunosensing. More specifically this invention demonstrates for the first time that LIG has been functionalized with antibodies for antibody-based electrochemical biosensing (immunosensing). Details regarding this major point is given below.
The laser inducing and biofunctionalization are amenable to scalable manufacturing. This is primarily due to the lack of need for preconcentration and labeling steps makes the biosensor low-cost and well-suited for one-time, disposable biosensing.
Other examples include electrochemical ion-sensing, pesticide monitoring, catalysis, and water-splitting, as will be discussed further herein.
Aspects of the invention include apparatus, methods, and systems utilizing functionalized LIG-based patterned electrodes for a variety of beneficial purposes.
One is sensors for electrochemical detection. Another is sensors for ionic species of interest. Another is sensors for monitoring for presence of a pesticide of interest. Another is energy generation by water splitting. An aspect of the invention is the combination of a specifically controlled laser for generation of a particular LIG pattern or patterns with effective use of the LIG pattern or patterns for a variety of purposes.
In the example of biosensing, including as an immunosensor, apparatus, methods, and systems according to the invention combine an LIG-based pattern which is effective for functionalization with relevant binding agents or anti-bodies, and then capture and measurement for target species or pathogens.
In the example of ion sensing, apparatus, methods, and systems according to the invention combine LIG patterns which are effective for functionalization to capture ionic species for detection and measurement.
In the example of pesticide monitoring, apparatus, methods, and systems according to the invention combine LIG patterns which are effective for functionalization to detect presence of a pesticide of interest in a sample or in situ. In the example of water-splitting, apparatus, methods, and systems according to the invention combine LIG patterns which are effective for functionalization as water-splitting electrodes.
Methods according to the invention include use of the foregoing apparatus for electrochemical detection. In addition, methods include direct or indirect writing of an effective LIG pattern(s) by selection and control of the operating parameters of a laser. Methods according to the invention including a combination of ordered steps to fabricate LIG electrodes and functionalize them as above.
Systems according to the invention include foregoing apparatus or methods in operative connection to other circuits or components. For example, electrochemical reading and analysis components are operatively connected to functionalized LIG electrodes according to aspects of the invention for use as bio-sensing, immunosensing, and pesticide monitoring. Electrical/electronic circuits and components are operatively connected to functionalized LIG electrodes according to aspects of the invention for ion sensing and water-splitting, The systems can include components for rapid and effective use on-site or for in-field sensing.
The following are incorporated by reference herein as if fully a part thereof and supplement this description at least as follows:
Background information about certain state-of-the-art practices by others can be found at the following, each of which is incorporated by reference herein: Cardoso, et al. Molecularly-imprinted chloramphenicol sensor with laser-induced graphene electrodes. Biosensors and Bioelectronics 124-125 (2019) 167-175; and Fenzl, et al. Laser-Scribed Graphene Electrodes for Aptamer-Based Biosensing. ACS Sens. 2017, 2, 616-620; and Hong et al. Direct-laser-writing of three-dimensional porous graphene frameworks on indium-tin oxide for sensitive electrochemical biosensing. Analyst, 2018, 143, 3327-3334.
Background information about functionalization of biosensors with binding agents can be found at the following, each of which is incorporated by reference herein: U.S. Patent Application Publication US2019/0330064 A1 (J. Tour, et al.); and U.S. Patent Application Publication US2020/00002174 A1 (J. Tour, et al.).
Background information about interdigitated electrodes fabrication, functionalization with antibodies, and sensing using a potentiostat can be found at the following, each of which is incorporated by reference herein: U.S. Pat. No. 5,958,791 (M. Roberts, et al.); and U.S. Patent Application Publication US2018/0059101 A1 (S. MacKay et al.).
For a better understanding of the invention and its aspects, non-limiting examples of some of the ways and forms those aspects can be made, used, and practiced will now be set forth in detail. It is to be understood these are examples only, and that these examples are neither inclusive nor exclusive of all forms and embodiments the invention can take.
These embodiments will be discussed primarily in the context of sensing of pathogenic bacteria in food samples. As will be appreciated by those skilled in this technical area, aspects of the invention can be applied in analogous ways to other biochemical analytes in non-food samples, for example, water quality, medical sensing applications, agricultural sensing applications.
These embodiments will be discussed primarily in the context of LIG electrodes. As will be appreciated by those skilled in this technical area, aspects of the invention can be applied in analogous ways to other patterns of LIG, for example, interdigitated electrodes (IDE), dipstick electrodes, serpentine electrodes, all-in-one electrodes (working, counter, and reference electrodes in the same device). As further discussed infra, aspects of the invention can be applied to a variety of working applications. These include but are not necessarily limited to electrochemical immunosensing, electrochemical ion sensing, pesticide monitoring, water-splitting, and electrochemical pesticide sensing, including neonicotinoids and others.
With reference to
As illustrated in
In some applications, pattern 14 is a single continuous pattern such as the dip-stick type pattern in
A more complex IDE pattern (see, e.g.,
The ability to focus and control in-plane movement of a laser 30 relative a surface 12 with very high resolution (at least μm scale features), provides tremendous versatility in creating patterns, whether one continuous pattern over a given part of a surface or multiple patterns over the same surface area. A subtle aspect of some embodiments of the invention is also control of the laser beam focus point out-of-plane, as will be described further herein.
The designer is provided with the ability to design and create such variety of patterns with available design and laser control systems. Thus, the scale of such LIG electrode-based apparatus according to the invention can vary from quite small (at least μm scale features and working areas) to larger if desired. As such, at the smaller end of scale and form factor, micro-scale individual sensors are possible or sets of micro-scale sensors. They can be easy to emplace for measurement, including in small volume samples. The LIG-pattern is functionalized appropriately for a given electrode application. The LIG-based pattern can take advantage of the well-known benefits of graphene, including physical, electrical, thermal, and other benefits.
Some specific examples focus on pathogen detection by emplacing relevant antibodies in the sensing portion of the LIG pattern. As will be appreciated, a variety of functionalizations are possible. Background information about functionalization with binding agents, including antibodies for immunosensing, can be found at U.S. Patent Application Publication US2019/0330064 A1 (J. Tour, and U.S. Patent Application Publication US2020/00002174 A1 (J. Tour, et al.), each of which is incorporated by reference herein. Other examples relate to other types of biochemical or electrochemical sensing. Others relate to electrode-based functionalities (e.g. water splitting).
In this example, a starting suitable substrate 12 which includes a carbon precursor is laser-scribed by spatial and power density control of laser 30 to generate a graphene pattern 14 of desired form factor. In one example, a dip-stick type working electrode pattern is scribed by laser 30, and has one portion 15 adapted for connection to a sensing circuit and the opposite end 16 adapted for functionalization as a sensing surface. In this case, a passivation material 17 can be overlaid pattern 14 between ends 15 and 16 to electrically insulate or block contact with that intermediate portion of pattern 14.
The result is an LIG electrode 10 with an exposed sensing surface or area 16 of porous graphene. Area 16 can be functionalized for a variety of electrode-based purposes, including electrochemical sensing purposes, electrochemical monitoring, and others, for example, water splitting. Examples of how LIG electrodes according to the invention can be functionalized for these varying applications will be discussed in detail infra.
In an example of electrochemical immunosensing illustrated in
When biofunctionalized end 16 (with immobilized antibodies 18) is exposed to a sample, if the sample contains a species of interest that binds to antibodies 18, by impedimetric techniques, measurements at surface 16 can detect (at ref. no. 30) the presence of the target 19 and, thus, functions as an immunosensor.
As will be appreciated, techniques to functionalize an electrode sensing area are well-known to those skilled in the art.
As such, the apparatus, methods, and systems of the generalized embodiments of
As will be appreciated, the invention can many forms and embodiments. It can also include ancillary or optional features. Some of those are discussed herein.
One example applicable to functionalization as an immunosensor is illustrated by
Other example of options, alternatives, and variations possible with aspects of the invention will become apparent herein.
Specific applications of aspects of the invention, and proof of concept data about them, are now set forth. As will be appreciated, these embodiments and examples meet at least some of the objects, features, advantages, and aspects of the invention.
With particular reference to
Foodborne illnesses are a growing concern for the food industry and consumers, with millions of cases reported every year. Consequently, there is a critical need to develop rapid, sensitive, and inexpensive techniques for pathogen detection in order to mitigate this problem. However, current pathogen detection strategies mainly include time-consuming laboratory methods and highly trained personnel. Electrochemical in-field biosensors offer a rapid, low-cost alternative to laboratory techniques, but the electrodes used in these biosensors require expensive nanomaterials to increase their sensitivity, such as noble metals (e.g., platinum, gold) or carbon nanomaterials (e.g., carbon nanotubes, or graphene). Herein, we report the fabrication of a highly sensitive and label-free laser-induced graphene (LIG) electrode that is subsequently functionalized with antibodies to electrochemically quantify the foodborne pathogen Salmonella enterica serovar Typhimurium. The LIG electrodes were produced by laser induction on polyimide film in ambient conditions, and hence circumvent the need for high-temperature, vacuum environment, and metal seed catalysts commonly associated with graphene-based electrodes fabricated via chemical vapor deposition processes. After functionalization with Salmonella-antibodies, the LIG biosensors were able to detect live Salmonella in chicken broth across a wide linear range (25 to 105 CFU mL−1) and with a low detection limit (13±7 CFU mL−1; n=3, mean±standard deviation). These results were acquired with an average response time of 22 minutes without the need for sample pre-concentration or redox labeling techniques. Moreover, these LIG immunosensors displayed high selectivity as demonstrated by non-significant response to other bacteria strains. These results demonstrate how LIG-based electrodes can be used for electrochemical immunosensing in general and, more specifically, could be used as a viable option for rapid, low-cost pathogen detection in food processing facilities before contaminated foods reach the consumer.
Nearly half a million people die each year from acquiring foodborne illnesses.1 This dismal statistic is only expected to increase as global food production and trade continue to rise to meet the demands of the increasing world population (over 9 billion by 2050 according to the United Nations prediction2). Hence, efficient food quality control measures are desperately needed to avoid wide-spread foodborne diseases and contamination. Data from Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) claim that one of the major contributors to foodborne illnesses is the bacteria Salmonella enterica, which causes about 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths in the United States every year.3,4 Furthermore, Salmonella causes an estimated $3.7 billion in economic burdens each year with exposure occurring through food, water, and contaminated surfaces.5 Despite strict regulations and efforts from producers to control pathogens in the food supply, growing numbers of foodborne illnesses are being reported globally.6
The reason for these illnesses is that contaminated food product (whether contaminated in the field or within food processing facilities) still passes unnoticed to the consumer. This is because foodborne pathogen detection is time-consuming and arduous. The gold standards for monitoring these pathogens include bacteria plate counting and polymerase chain reaction (PCR) experiments that may take several days due to pre-enrichment steps and necessary laboratory processing.7 Hence, all food products passing through the doors of food processing facilities are not tested for pathogens as some food would spoil before tests could be performed and most food products have low/tight profit margins making ubiquitous testing infeasible.8,9 Therefore, there is an urgent need to create a rapid (less than 1 hour), low-cost (less than $1), and highly sensitive (detection limits comparable to plate counting and PCR methods) sensor systems that can be used on-site to detect foodborne pathogens such as Salmonella.10,11 Recent research into electrochemical biosensors, including our own,12-16 has demonstrated promising potential for such in-field pathogen and containment detection.17-19
Electrochemical biosensors have been explored extensively in recent years as an alternative to conventional methods for detection of pathogenic bacteria, mainly due to their high sensitivity, easy handling, fast response time, and low costs.20-23 Moreover, comparing them with other commonly used techniques, such as colorimetric and fluorescence assays, electrochemical transducers have significant advantages since they do not require laborious interpretation and equipment resources, exhibit more versatile detection schemes which provide broader applications, and are capable of real-time quantification.24,25 Also, electrochemical biosensors have received particular attention since they can perform direct and label-free measurements, and can be easily manipulated by personnel without previous training (e.g., home glucose monitors for diabetics26,27). Moreover, electrochemical biosensors that are modified with carbon nanomaterials such as graphene have significantly improved biosensor performance28,29 and have increasingly been applied to food safety and sustainable agriculture applications.13,14,30 Recent reports have demonstrated potential in developing sensitive and accurate electrochemical Salmonella detection platforms.24,31-33 However, some of these biosensors are complex and costly because they require expensive (noble metals34,35) or difficult to fabricate materials (e.g., gold nanoparticles biofunctionalized with enzymes,36 nanocomposites using graphene oxide and titanium isopropoxide37) to improve signal amplification and/or complex manufacturing steps (e.g., cleanroom processing38). It should also be noted that the main challenge in the field of biosensing for monitoring pathogens is not the response time, but the poor detection limit, approximately 102-103 CFU mL−1 (detection limits of ≤5 CFU mL−1 are required for ensuring pathogen-free food products10,17). To overcome this hurdle, most studies have integrated a pre-concentration step, which improves detection limit, but obfuscates the rationale for creating the rapid sensor in the first place. However, a graphene biosensor may help to overcome these detection limit shortcomings by improving the sensor sensitivity.
Within the category of two-dimension materials, graphene is considered outstanding due to its structure and exceptional properties.39,40 Graphene is a sheet of sp2 bonded carbon atoms arranged into a rigid hexagonal lattice, exhibiting a set of properties that no material has concomitantly displayed; for example, high mechanical strength (1012 Pa), excellent electrical conductivity and charge carrier mobility (˜105 cm2V−1s−1) large specific surface area (˜2630 m2g−1), and high impermeability and biocompatibility.41-43 Consequently, graphene-like nanomaterials have attracted attention as emerging materials for electrochemical sensor applications.44 Techniques for graphene electrode fabrication have grown considerably to supply the demand for this material;39 however, common methods of synthesis, such as photolithography45,46 (an expensive cleanroom processing technique), chemical vapor deposition (CVD),47 laser ablation48 include high thermal requirements, low-pressure (vacuum) requirements, or multiple steps towards chemical formation of graphene.49 Moreover, post-synthesis processing is generally required to transfer the graphene to a non-conductive substrate50 which further increases the time and cost of electrode fabrication. An alternative to these expensive graphene electrode fabrication techniques is to produce sensors based on direct-write processes such as inkjet printing or aerosol jet printing that are capable of printing graphene electrodes from graphene flakes synthesized from bulk chemical exfoliation of graphite.51-54 Though these graphene-electrode fabrication methods do not retain the high-performance characteristics of pristine CVD-grown graphene, for example, they do display sufficient electrical conductivity and biocompatibility needed for a variety of sensor applications and eliminate the high cost of alternative graphene synthesis protocols and graphene transfer methods. However, these printing techniques often require additional post-print processing (e.g., laser,53 thermal,55 or photonic annealing56) to increase the electrical conductivity of the printed graphene which further complicates their fabrication process.
As an alternative to these techniques, the Tour group49 introduced a simple one-step, direct-write graphene electrode fabrication method, called laser-induced graphene (LIG). LIG is typically formed by converting sp3 carbon found in polyimide into highly conductive sp2 hybridized carbon found in graphene through CO2 laser induction57,58 (though some have demonstrated LIG formation on polyimide using a UV laser59,60). LIG combines both the graphene synthesis and graphene electrode fabrication steps into one simple process, using a laser to selectively convert distinct patterns of polyimide into a high-surface graphene circuit that is often nano/microstructured or porous. Since LIG can be easily manufactured from commercial polymers, it has been applied towards stretchable and sensitive strain gauges,61 non-biofouling surfaces,62 microsupercapacitors,63 UV photodetectors,64 sound generators and detectors,65 and more recently, electrochemical sensors.66 Recently, an electrochemical biosensor based on LIG-electrodes was developed for the detection of biogenic amities in food samples;59 similarly, in another study an electrochemical biosensor was developed based on LIG that showed the ability to detect low levels of the antibiotic chloramphenicol, which is banned in food production.35 Another example is the electrochemical LIG-sensor used for fouling-biofilm detection, one of the main challenges in the food industry,62 while another LIG sensor was capable of monitoring the concentration of nitrogen (both ammonium and nitrate ions in soil solutions) in the hopes of better monitoring and controlling fertilizer inputs in farm fields to maximize crop yield while lowering fertilizer waterway pollution due to excess fertilizer use.60 However, electrochemical pathogen sensing using LIG has yet to be demonstrated.
Herein, we report on the first LIG sensor that is capable of rapid and quantifiable detection of Salmonella enterica concentrations in food samples. Porous graphene was produced from polyimide by laser induction, and then characterized conferring a new potential application in the sensing field. An impedimetric immunosensor was developed based on LIG-electrodes functionalized with specific antibodies for detection of Salmonella enterica, one of the most prominent foodborne pathogens.67 The immunosensor was able to detect the pathogen at low concentration, 13±7 CFU mL−1 in complex media, chicken broth, with a response time of 22 minutes. Electrochemical impedance spectroscopy was used as a label-free detection over a broad range of bacteria concentrations, from 25 to 105 CFU mL−1. Moreover, this promising device is a low-cost and disposable sensor that can be used in-field or at the point-of-service (e.g., food processing facilities) for the detection of contamination, which reinforces its important contribution to food safety.
Polyimide (Kapton, 0.07 mm) tape was purchased from McMaster-Carr co. (Elmherst, Ill., USA), and Epson Ultra Premium Photo Luster (240 g m−2) was acquired from Office Depot (Boca Raton, Fla., USA). Potassium ferro/ferricyanide, N-Hydroxysuccinimide (NHS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 2-(N-morpholino) ethanesulfonic acid (MES), and ethanolamine were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Tryptic soy agar (TSA), tryptic soy broth (TSB), tryptose phosphate broth (TPB), and buffered peptone water (BPW) were purchased from Criterion Dehydrated Culture Media (Hardy Diagnostics, Santa Maria, Calif., USA). Potassium chloride and SuperBlock™ in phosphate buffered saline (PBS) (used as blocking buffer) were purchased from ThermoFisher Scientific (Waltham, Mass., USA). KPL BacTrace polyclonal antibody anti-Salmonella was purchased from SeraCare (USA). PBS was purchased from Alpha Aesar (Tewksbury, Mass., USA), and chicken broth was purchased from a local supermarket. All the chemicals used in this study were analytical grade. Solutions were made using deionized with an electric resistance of approximately 18.2 MΩ.
The working electrode was designed using a linear sketch pattern (0.17 mm separation) in SolidWorks 2018 (Dassault Systems, France), and the engraving process was performed with a 75 W Epilog Fusion M2 CO2 laser (Epilog Laser, Golden, Colo., USA) at 7% speed and 4% power with a lens to material distance of ˜74 mm and beam size of ˜176 μm in ambient atmosphere. The laser induction was carried out on polyimide film taped onto the emulsion side of the photo paper, as previously described by Tehrani and Bavarian,68 and Fenzl et al.69 This procedure 40 produced LIG-electrodes as shown in
The Raman spectrum was obtained by using a Renishaw InVia confocal Raman microscope with a 633-nm laser source (0.12 mW), a 50× objective lens and a diffraction grating of 1800 lines, in order to confirm the graphene formation by the laser induction process. The crystallinity of the bare electrodes and the level of graphitization were evaluated using a Bruker D8 DISCOVER X-ray Diffractometer provided with copper radiation (λ=1.542 Å) scanning θ/2θ. A scanning electron microscope (SEM) JEOL JSM-6010LA equipped with an energy dispersive spectroscopy (EDS) system was used to obtain images of the LIG morphology at 230×, 2000×, and 2300× magnification, and the electrode's chemical composition, at accelerating voltage of 5 kV.
Antibody functionalization onto LIG-Electrodes
To determine the optimum concentration of polyclonal antibody anti-Salmonella to functionalize the LIG-electrodes, different concentrations of antibody were initially functionalized on the electrode surface in an effort to maximize immunosensor performance. Briefly, the working area 16 of the electrodes 10 was covered with 30 μL of EDC/NHS (3:1) solubilized in sterile filtered MES (pH 6.0) for 1 hour and then rinsed with 1× dilution of PBS (1×PBS) pH 7.4 to remove the unreacted EDC/NHS. Next, polyclonal antibody 18 anti-Salmonella at different concentrations (0.5, 1.0 and 1.5 μM) was applied to the surface of the working electrode 10 followed by overnight incubation at 4° C. See method 50 at
The electrochemical proprieties of the LIG-electrodes 10 were analyzed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). All electrochemical measurements were carried out on a CH Instruments Electrochemical Analyzer (CHI7081E model, CH Instruments, Inc., Austin, Tex., USA) at room temperature. The 3-electrode system consisted of a CH Instruments Ag/AgCl reference electrode, platinum counter electrode, and the LIG as the working electrode 10. CV and EIS experiments were carried out in 10 mL solution containing 0.1 M KCl, 4 mM K3[Fe(CN)6], and 4 mM K4[Fe(CN)6]. The scan rates used for CV measurements were 50; 75; 100; 125; 150; 175; 200 mV s−1, in a sweep range from −0.4 V to 0.6 V with a quiet time of 2 seconds between sweeps. The average sheet resistance, n=3, was taken at ambient conditions (25° C.) on a Variable Temperature Hall Effect Measurement System (Model H5000, MMR Technologies, San Jose, Calif., USA). EIS analyses were performed in the frequency range of 1 MHz 100 Hz, using AC amplitude of 10 mV and DC voltage of 0 V.
Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028), Bacillus cereus (ATCC 14579), Escherichia coli O157:H7 (ATCC 43895), Listeria monocytogenes (ATCC 15313), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus aureus (ATCC 29213) were used to test the immunosensor 20. Bacteria strains stored at −80° C. were resuscitated through 2 consecutives 24 h growth cycles in TSB at 35° C. L. monocytogenes was resuscitated under the same time and temperature conditions in TPB. B. cereus was also resuscitated twice in TSB for 24 h but at 30° C. Bacteria cultures were renewed weekly in TSB or TPB (i.e., one transfer followed by 24 h incubation in aerobic conditions) and maintained at 4° C. Samples of bacteria were serially diluted in BPW, plated via spread plating on TSA and incubated for 18 hours at 35° C. or 30° C. before counting the colony growth, and results were reported as CFU mL−1. Different bacteria concentrations, ranging from approximately 25 to 107 CFU mL−1, were prepared in 15 mL of BPW or chicken broth in order to evaluate the impedimetric immunosensor 20 and to simulate its application in food. Plate counting was used parallelly to the immunosensing experiments to confirm the concentration of the bacterial dilutions and validate the impedimetric results.
The presence of bacteria was evaluated by EIS analysis, measuring different bacteria concentrations directly in suspension with incubation time of 20 minutes under 180 rpm stirring and analysis time of 90 seconds. Before testing the immunosensor in complex media, its performance was verified in pristine buffer, BPW. Between each measurement, the electrode was thoroughly washed with ix PBS to remove unbound bacteria. Complex plane diagrams (Nyquist plots) were used to determine the charge transfer resistance (Rct), the solution resistance (Rs), the double-layer capacitance (Cdl), and the Warburg element37 (Zw), fitting the EIS data sets to an equivalent circuit model (i.e., Randles-Ershler circuit) through EIS Spectrum Analyser from ABC Chemistry (Minsk, Belarus). It should be noted that the diameter of the semicircle obtained from Nyquist plots is a measure of the charge transfer resistance (Rd) used to calibrate the concentration of Salmonella attached to the developed biosensor as explained in greater detail in the Results and Discussion section. The LIG-based immunosensor 20 was also evaluated through a selectivity test using the following five foodborne pathogens 19: Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes. These bacteria were chosen due to their importance to food safety and were tested under the same conditions used for Salmonella enterica at a constant concentration of 104 CFU mL−1.
The measurements were made in triplicate and results were expressed as mean=1, standard deviation. Differences between variables were tested for significance using one-way analysis of variance (ANOVA) and significantly different means (p<0.05) were designated using Tukey's Honestly Significant Differences (HSD) test through IMP v.13 Software (SAS Institute, Cary, N.C., USA). The functional correspondence among quantitative variables was performed using SigmaPlot 12 (Systat, San Jose, Calif., USA) by regression analysis. To evaluate the electroactive surface area (ESA) and the heterogeneous electron transfer rate (HET), the peak current values and peak potential separation from the CV results were used to solve the Randles-Sevick equation59,66 and to apply the Nicholson method for reversible electron transfers,70 respectively. The 3G method was used to calculate the limit of detection (LOD) and sensivity.59,71 Please, see Supporting Information for further details on calculations and data presentation and analysis.
First, SEM was used to characterize the surface topography of the LIG electrodes 10 (
Next, EDS, Raman spectroscopy, and X-Ray Diffraction (XRD) were performed to analyze the structure of the materials, as well as the surface molecular groups on the LIG electrode 10. The C—O, C—N and C═O bonds originally present in polyimide film 12 could easily be broken by the high temperature,49 as confirmed by EDS (
Raman spectroscopy was used to determine the graphitic properties of LIG. This technique is also useful to characterize disorder in the resultant sp2 carbon lattice.43 The Raman spectrum showed three main peaks displayed in
The electrochemical performance of the bare LIG-electrodes 10 was investigated in order to verify its ability to act as an electrochemical transducer. CV curves were recorded and for all scan rates tested the electrodes displayed well-defined redox peaks (
The CV curves also convey information about the heterogeneous electron transfer rate (HET) between the electrode 10 and the redox mediator species.66 The HET constant obtained (k0=0.0146±0.0031 cm s−1) exceeds those found by other groups59,69,78 ranging from 0.0030 to 0.0044 cm s−1 for LIG with the same redox ferro/ferricyanide species. It also exceeds commercial edge plane pyrolytic graphite (0.0026 cm s−1) and basal plane pyrolytic graphite (0.0003 cm s−1), as reported in a previous study by Griffiths et al.77 These results confirm the effective electron transfer kinetics of LIG produced in this study, and its subsequent feasibility for use as an electrochemical transducer. Furthermore, the average sheet resistance of the LIG-electrodes 10 was 12.7±1.6 kΩ sq−1, which is significantly lower than previously reported values of LIG based electrodes (15-20 kΩ sq−1),60 and also lower than electrodes based on inkjet-printed graphene with reported sheet resistance of 34 kΩ sq−1.52 Thus, the results obtained from CV, EIS and sheet resistance confirm that the LIG-electrode 10 fabricated in this study is suitable for electrochemical sensing.
The bare LIG-electrode 10 was converted into an immunosensor 20 by functionalizing the surface with polyclonal antibodies 18 to detect Salmonella enterica Typhimurium 19 via carbodiimide cross-linking (see methods), as shown in
The presence of attached bacteria cells 19 plays the role of electron kinetic barrier as well as steric hinderance,20 decreasing the electron transfer path between the electrolyte solution and the electrode, and consequently resulting in the increase of Rct values. A calibration plot was obtained by normalizing the Rct with respect to the Rct value measured for zero concentration of Salmonella enterica in the buffer solution. The LIG-based immunosensor 20 presented a linear sensing range from 25 to 103 CFU mL−1 (R2=0.984), with sensitivity of 42Ω log CFU−1 mL and a limit of detection of 10 CFU mL−1 in buffer (
Further, the LIG-based immunosensor was tested for selectivity using 5 different bacteria strains under the same conditions as those used for Salmonella enterica in chicken broth at 104 CFU mL−1. The Rct values recorded from interference testing did not show significant change among the bacteria tested and presented an average value of 4.8% for the ΔRct (
The shelf life of freeze-dried immunosensors was evaluated during 7 days of storage at −20° C. (see Supporting information for details). As it can be observed in
The developed immunosensor exhibited overall good performance using easily obtainable and inexpensive materials with an estimated materials cost of $1.76 per immunosensor (approximate cost breakdown: polyimide=$0.15, EDC-NHS=$0.01, Superblock=$0.03, ethanolamine=$0.04, antibodies=$1.53), which contributes to its accessible fabrication. Table 1 (below) summarizes the performance characteristics of the immunosensor prepared in this work, as well as other similar biosensors in the recent literature. Previous studies have developed highly sensitive and label-free Salmonella spp. sensors, for example sensors reported by Silva et al.67 and Punbusayakul et al.82 based on ion selective electrodes made of gold nanoparticles and double-walled carbon nanotubes, respectively, but all require an hour to multiple hours to obtain a signal which is longer than 22 minutes, the response time reported herein. Moreover, biosensors that displayed performance similar to this work used expensive materials and/or complex fabrication methods, such as gold67,83 or required multi-step fabrication to develop the electrodes.84 Furthermore, the sensitivity reached by the immunosensor developed herein was significantly higher than other recent graphene-based sensors, even in complex matrices, with a limit of detection 2× lower than the one obtained by Jia et al.84 and 7× lower than the one obtained by Fei et al.85 Thiha et al.86 and Appaturi et al.87 report impressive analysis times (10 minutes and 5 minutes, respectively). However, both report the necessity of various pieces of laboratory equipment and chemicals leading to a much more complex and longer fabrication process than reported in this work. Since this work reports a process that requires only a Laser and a polyimide substrate for electrode fabrication, it has the advantage of easier upscaling for mass production. These devices also use sample volumes of 5 μL and 1 mL, respectively, which might require sample preconcentration steps to avoid false negatives and consequently increase test response time. Based on the performance characteristics shown in Table 1 (below), there are no concomitant records of a rapid (22 minutes or less), label-free, sensitive, and simple to fabricate sensor similar to the one demonstrated in this work that can selectively detect Salmonella enterica from 25 to 105 CFU mL−1, which covers relevant levels for food safety analysis.
This work reports on a highly sensitive, selective, and easily fabricated impedimetric immunosensor by direct formation of graphene on commercial polyimide film through a laser induction technique. The results obtained reinforce that this sensor can be widely implemented due to its simple fabrication protocols with equipment that is accessible throughout the world. This immunosensor is a versatile device that could be distinctly functionalized for monitoring other pathogens besides Salmonella enterica Typhimurium, depending on the selectivity of the biorecognition agent. The working electrode based on LIG displayed a high ESA and HET with values of 0.104 cm2 and 0.0146 cm s−1, respectively, and was functionalized with antibodies for Salmonella enterica detection. The immunosensor presented a limit of detection to the target bacteria of 13±7 CFU mL−1 in complex media, chicken broth, in just 22 minutes without any pre-treatment. In addition, the sensor exhibited a wide linear sensing range, from 25 to 105 CFU mL−1. Therefore, impedimetric immunosensors based on LIG are very promising for bacteria sensing, since it is easily manufactured in ambient conditions compared to other complex fabrication procedures that require CVD76 and/or sophisticated substrate-transfer techniques, ink and ink-preparation52 or post-printing processes.53 Consequently, resulting in a low-cost fabrication process that produces porous graphene with high electrical conductivity and chemical stability.88 All of these properties demonstrate that the developed biosensor is well-suited for use in food safety monitoring and, in general, a platform that could be modified with different biorecognition agents for future electrochemical biosensors.
Supporting information is at https://pubs.acs.org/doi/10.1021/acssensors.9b02345.
37
67
20
23
34
89
31
82
85
75-105
84
90
83
86
87
It can therefore be seen that the foregoing Specific Embodiment 1 meets or exceeds at least one or more of the objects, features, advantages, and aspects of the invention. This example includes proof-of-concept data regarding this embodiment and its examples.
With particular reference to
Laser induced graphene (LIG) has shown to be a scalable manufacturing route to create graphene electrodes that overcomes the expense associated with conventional graphene electrode fabrication. Herein we expand upon Specific Example 1, supra, by functionalizing the LIG with metallic nanoparticles for ion sensing (Specific Example 2), pesticide monitoring (Specific Example 3), and water splitting (Specific Example 4). The LIG electrodes were converted into ion-selective sensors by functionalization with poly(vinyl chloride)-based membranes containing K+ and H+ ionophores. These ion selective sensors exhibited a rapid response time (10-15 s), near-Nernstian sensitivity (53.0 mV/dec for K+ sensor and −56.6 mV/pH for pH sensor), long storage stability for 40 days, and were capable of ion monitoring in artificial urine. The pesticide biosensors were created by functionalizing the LIG electrodes with the enzyme horseradish peroxidase and displayed a high sensitivity to atrazine (28.9 mA/μM) with negligible inference from other commons herbicides (glyphosate, dicamba and 2,4-dichlorophenoxyacetic acid). Finally, the LIG electrodes also exhibited small overpotential for hydrogen evolution reaction and oxygen evolution reaction. The OER tests yielded overpotentials of 448 my and 995 mV for 10 mA/cm2 and 100 mA/cm2, respectively. The HER tests yielded 35 mV and 281 mV for the corresponding current densities. Such versatile LIG platform paves the way for simple, efficient electrochemical sensing and energy harvesting applications.
Graphene is a one-atom-thick sheet of carbon atoms arranged in a honeycomb-like structure. It is characterized by large surface area, high electrical conductivity, high mechanical stiffness and thermal conductivity1. Such properties make graphene-based electrodes particularly attractive for various electrochemical applications, including ion sensing, amperometric sensing, and water splitting2,3. Traditional methods for the creation of graphene-based materials generally includes chemical vapor deposition (CVD) which is a laborious process that requires vacuum chambers and high temperatures (500-1000° C.)4. Furthermore, after such CVD synthesis, additional complex procedures is often required to transfer the synthesized graphene to insulating dielectric materials that are suitable for electronic devices5,6. To simplify graphene-based circuit fabrication, researchers have developed a variety of printing techniques including inkjet7,8, screen9, and dispenser printing10. Though these techniques generally print exfoliated flakes of graphite (graphene or graphene oxide) that are less conductive then pristine CVD-grown graphene, researchers have developed a variety of post-print annealing techniques (i.e., rapid pulse laser annealing, high-temperature thermal annealing, and photonic annealing) that improves the electrical conductivity to levels near printed metals (<1k Ω)7,11-13. Recently researchers have been able to effectively combine direct write printing and post-print annealing to create highly conductive graphene circuits via a fabrication method coined laser-induced graphene (LIG) or laser scribed graphene (LSG)14-17. LIG can be produced from carbon-based polymers (i.e., polyimide) by direct write CO2 laser irradiation which converts the hybridization of carbon within the polymer from sp3 to sp2 found in graphene15. LIG circumvents the need for ink formulation and post-print annealing involved in graphene printing techniques, uses inexpensive raw materials, and can create electrode designs on-the-fly as circuits can be rapidly created with CAD software and uploaded into the CO2 laser.
Researchers have been able to functionalize the surface of LIG with nanoparticles and/or biorecognition agents to create a wide variety of electrodes which include those capable of splitting water and water oxidation18,19, sensing fertilizer and hydrations ions in soil and sweat respectively20,21, monitoring of biogenic amines and Salmonella bacteria in food samples16,22, impedance-based cell monitoring23, aptamer-based biosensing in serum17, and biomarkers in sweat24. In this section, we will expand upon these reports by demonstrating a simpler approach to creating LIG electrodes for water splitting, expanding the research of LIG-based ion sensing to include pH monitoring, and introducing the first example of a pesticide monitoring LIG-based electrode through functionalization with an enzyme.
Potentiometric ISEs have been used for the selective and sensitive (nanomolar detection limits) detection of ions in a wide variety of applications including detecting perchlorate and iodide in water25,26, ammonium and potassium in urine27, and lithium in whole blood28,29. Conventional liquid-junction ISEs contain an aqueous internal reference electrolyte between the working electrode and ion selective membrane that requires maintenance and creates long-term stability due to leeching. However, solid-contact or solid-state ISEs do not contain such a liquid reference electrolyte which can improve their stability and decrease their maintenance30. In the present work, we develop solid-state ISEs for the detection of K+ and pH (pH ISE is in fact a sensor for H+). These targets were selected since their detection plays an important role in environment sensing31, human health monitoring27, and medical diagnostics (e.g. for use on catheter tips or in implantable devices)32. The created K+ and pH ISEs have a wide linear range (0.3-150 mM K+ and pH 5-8), near-Nernstian sensitivity (53.0 mV/dec for K+ ISE and −56.6 mV/pH for pH ISE), and excellent storage stability during at least 40 days.
In addition to ion sensing, we demonstrate how LIG-based electrodes can be used for pesticide monitoring through functionalization with the enzyme horseradish peroxidase (HRP). Horseradish peroxidase (IMP), a helve group enzyme has shown selective measurement of hydrogen peroxide through amperometry33,34. Electrodes functionalized with HRP have been used to monitor a wide variety of analytes including glyphosate, atrazine, and dichlofenthion35-37. However, we demonstrate that LIG electrodes functionalized with HRP can selectively monitor the herbicide atrazine with high sensitivity (28.9 mA/μM) and report minimal interference from other herbicides commonly applied in the Corn Belt region of the United States including glyphosate, dicamba, and 2,4-dichlorophenoxyacetic acid.
Finally, we demonstrate how LIG-based electrodes can be used in electrochemical energy conversion. Electrochemical electrolysis has gained recent attraction due to its ability to increase hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) kinetics38-40. To date, various noble metals and semiconductors have been promising as HER catalysts. Phosphides41,42, sulfides, and manganese has shown good performance in HER. Hydroxides/oxides of nickel43, iron43,44, cobalt45 has shown good performance in OER. However, metallic platinum (Pt) remains as one of the most promising used catalysts for electrolysis due to its high catalytic activity in liberating H2 at high reaction rates and low overpotentials (η). To reduce the cost but maintain the superior catalytic activity, nanoscale amounts of platinum in the form platinum nanoparticles (PtNPs) are often deposited onto the electrodes via drop-cast46, thermal decomposition of Pt salt18,47, and atomic layer deposition48. However, recently researchers have demonstrated that electroless deposition of PtNPs is a simple one-pot synthesis process offering several advantages compared to commonly used methods to synthesis electrocatalysts (such as solid-phase reaction and hydrothermal, which require delicate control of reaction time and temperature. The developed LIG-Pt and LIG-NiO electrodes display a high overpotential 100 mA/cm2 for HER, and OER is as low as 281 and 995 mV with relatively low Tafel slopes of 82 and 48 mV/dec, respectively.
The LIG electrodes were prepared by irradiation of polyimide (0.125 mm thick, DuPont, USA) by the CO2 laser (75-watt Epilog Fusion M2, USA). The polyimide was fixed on a glass substrate and cleaned with a wipe before lasing. Drawings of the electrodes were prepared in CorelDraw and then submitted to the laser-controlling program. Lasing parameters were the following: 7% speed, 7% power, 50% frequency, raster mode, 600 dpi, and all other parameters were selected as “off”. The laser beam was defocused by the placement of polyimide sheet 2 mm lower than the focus distance. These conditions were experimentally selected to achieve the highest quality of graphene.
Preparation of LIG-NiO electrodes. Once LIG electrodes were lasered, 15 μL of 1M nickel (II) acetate tetrahydrate was deposited onto each electrode. The solution was allowed to dry for 10 minutes. The CO2 laser, running the same etching pattern, lasered the LIG a second cycle to induce the thermal decomposition of the nickel salt into nickel (II) oxide and other gases, such as oxygenates, hydrogen with hydrocarbons, and carbon oxides. The silver paste was applied to the stem of the LIG electrode. Acrylic polish was applied to the electrode to ensure a constant, working surface area of 25 mm2 square throughout experiments.
Preparation of LIG-Pt electrodes. The deposition of Pt on LIG is followed by the recipe reported previously49-52. A chloroplatinic/formic acid solution with a 100:5 ratio, respectively, was used to electrolessly deposit Pt onto bare LIG electrodes. Bare electrodes were placed into a glass vial containing 10 mL, of the acid solution. The pH of the solution was changed from 1.68 to 1.75 by adding Ammonium Hydroxide in order to increase the rate of Pt deposition. The electrodes sat in the solution for 24 hours under ambient conditions. In a similar manner to the LIG-NiO electrode, silver paste and acrylic polish were applied.
Mixture for the preparation of K+ ISE contained valinomycin (potassium-selective ionophore, 2.5 mg), dioctyl sebacate (plasticizer, 165 mg), poly(vinyl chloride) high molecular weight (the main solid component of the membrane, 82.5 mg), and tetrahydrofuran (solvent, 1667 mg)53. Mixture for the preparation of pH ISE contained hydrogen ionophore I (5 mg), potassium tetrakis(4-chlorophenyl)borate (ionic additive for better ion exchange, 2.5 mg), dioctyl sebacate (327.5 mg), poly(vinyl chloride) high molecular weight (165 mg), and tetrahydrofuran (3163 mg)54. After preparation, the mixtures were vortexed for 3 min, left in the fridge (+4° C.) overnight for a complete dissolution of the components and vortexed again for 3 min before usage. The mixtures could be used for several months if stored in the fridge. However, to achieve the best sensor performance it is better to prepare new mixtures every 2-3 weeks. K+ and pH ISEs were prepared by drop-casting of 8 μL of the corresponding ionophore mixture onto the terminal circular part of the electrode. About 1 mm of the surrounding area (polyimide) was also covered with the mixture to prevent direct contact between the tested solution and the graphene. For the same reason, the central part of the electrode was covered in advance with acrylic polish. The prepared ISEs were stored dry at room temperature.
Preparation of Horseradish Peroxidase—LIG electrodes. Once LIG electrodes were lasered, acrylic polish was applied to the stem of the electrode to act as a passivation layer. Silver paste was applied to the contact of the electrode. A solution of 3% Horseradish Peroxidase (HRP) Type VI (Sigma Aldrich) and 2% Glutaraldehyde (weight) was prepared fresh in a 1:1 volumetric ratio. After mixing this solution, 1 μL of the mixture was drop-coated onto the pad of the working electrode. The deposited enzyme mixture on the electrode was allowed to dry for 12 hours at 4° C. before testing.
Artificial urine for testing of K+ and pH ISEs was prepared according to55, with few modifications. The artificial urine was obtained by dissolving the following components in DI: sodium L-lactate (1.1 mM), sodium citrate dehydrate (2 mM), sodium bicarbonate (25 mM), urea (170 mM), uric acid (0.4 mM), calcium chloride (2.5 mM), sodium chloride (90 mM), magnesium sulphate (2 mM), sodium sulphate (10 mM), sodium dihydrogen phosphate (7 mM), disodium hydrogen phosphate (7 mM). The solution was filtered through a 45 μM syringe filter to remove larger undissolved components. Thus, all ions found endogenously in urine were present in the artificial urine except potassium and ammonium. The pH of the as-prepared artificial urine was 8.5 and it was decreased to lower values (pH 5, 6, 6.5, 7, 7.5, 8) using hydrochloric acid (HCl).
Water splitting electrodes. A three-electrode setup was used on a CHI 6273E electrochemical analyzer (potentiostat), with a Pt wire as the counter electrode, Ag/AgCl as the reference electrode, and LIG as the working electrode. The tests were performed in 1M KOH. Linear Sweep Voltammetry (LSV) was performed for both the LIG-NiO and LIG-Pt electrodes. The LIG-Ni electrode was tested for the Oxygen Evolution Reaction (OER) from 0 to 1.7 V (vs Ag/AgCl) at a scan rate of 5 mV/s. The LIG-Pt electrode was tested for the Hydrogen Evolution Reaction (HER) from 0 to −1.7V (vs Ag/AgCl) at a scan rate of 5 mV/s.
Ion-selective electrodes. The developed LIG ISEs were electrochemically characterized with a CHI 6273E electrochemical analyzer (potentiostat). The potentiostat operated in “open circuit potentiometry” mode, with 0.5 s interval between separate readings. An ISE was connected to the working connector, and a reference electrode (CHI111 Ag/AgCl liquid-junction reference electrode filled with 1 M KCl) was connected to combined reference and counter connectors. The ISE and the reference electrode were placed in a 10 mL glass beaker filled with 4 mL of DL with constant slow stirring (˜100 RPM) by a magnetic stirrer. For the sensor calibration, aliquots of concentrated KCl solution (1, 10, 100, and 2000 mM in DI) were added to the working cell. Calibration of pH ISEs was carried out in standard pH solutions with pH 4, 7, and 10 (Fisher Scientific) and in 1 mM phosphate buffer with different pH levels adjusted with HCl and NaOH.
Horseradish Peroxidase—LIG electrodes. A three-electrode setup was used on a CHI 6273E electrochemical analyzer with a platinum wire counter electrode, Ag/AgCl reference electrode, and functionalized LIG working electrode. Amperometric i-t tests were performed at −0.1 V. The test cell contained phosphate buffer saline pH 7.4. A calibration experiment for atrazine was conducted by starting the i-t test. Upon reaching steady state, 0.4 mM hydrogen peroxide was added to the test cell. As the oxidation-reduction reaction occurred between HRP and H2O2, a signal was detected. Once this signal reached steady state, atrazine was added in 10 μM increments, allowing a steady state signal to be reached before the next addition. The test cell contained PBS (pH 7.4). The same three-electrode setup was used. An i-t test was performed for a single sensor by testing the response to each pesticide at 10 μM additions. The test setup was rinsed 2 times with PBS (pH 7.4) at the end of every test. The same sensor and procedure were used to test for the detection of dicamba, 2,4-dichlorophenoxyacetic acid, and glyphosate.
Microscopy Characterization Microscopic images are taken from a FEI Quanta 250 FEG Scanning Electron Microscope (SEM) at an operating voltage of 10 kV with EDS. An Oxfords Instruments Aztec X-Max 80 detector system connected to FEI Quanta 250 was used to collect the EDX photons for elemental detection. Raman spectrum was obtained by using an XploRa Plus confocal Raman upright microscope equipped with a 532 nm excitation source and a Synapse Electron Multiplying Charge Coupled Device camera (Horiba Scientific/NJ, France). The intensity (height) was determined for the D, G, and 2D bands by fitting the data to a Lorentzian model. XPS measurements were performed using a PHI ESCA 5500 instrument. The sample was irradiated with 200 W unmonochromated Al Kα X-rays. CasaXPS was used to process raw data files.
The LIG electrodes 10B and 10C used for these tests were produced by etching 0.005″ polyimide (PI) with a CO2 laser 30 as shown in
Scanning electron microscopy (SEM) images with corresponding EDS were acquired to investigate the morphology of the LIG electrodes. The pristine LIG 10 (
Pt was electrolessly deposited onto the LIG by immersing the sample into chloroplatinic acid solution following the protocol that was previous developed in our group49-51. The method can deposit platinum onto various surface such as carbon based (carbon nanotube, carbonized wood, graphene) and others like cellulose and Sift wools with surface functionalization50-52. After about 20 hrs, a layer of Pt is deposited onto the surface of LIG as shown in
Electrochemical performance of LIG were characterized by cyclic voltammetry in 5 mM ferricyanide-ferrocyanide mixture. Eleven electrodes from the same batch were tested (
Other advantages of the developed LIG electrodes are fast and easy production process (it takes 12-15 s to lase one electrode) and low cost of materials (cost of polyimide sheet for one electrode is $0.02). For comparison, commercial CVD-grown graphene film (1×1 cm2, 4 items) cost $352 (Sigma-Aldrich, ref. #773719). Recently reported electrode based on carbon nanotubes, characterized by the authors as simple and fast in production, requires 5-step preparation process that lasts ˜6-8 h62. Graphene modified with MoS2 nanocrystals was synthesized 45-second microwave annealing of reaction mixture (MoCl5, graphene oxide, thiourea, and ethanol) using conventional microwave oven63.
Calibration of K+ ISEs was carried out in DI by sequential increase of KCl concentration in the working cell (
Typical calibration curve of the K+ ISE is shown on
Storage stability of the sensors was also investigated. For this experiment K+ ISEs were calibrated after preparation and after 40 days of dry storage at room temperature (20-22° C.). Sensitivity of the sensors remained the same, which indicates excellent storage stability. The characteristics of the developed K+ ISE are similar to other solid-state electrodes found in literature. For example, solid-state K+ ISE based on graphene-covered paper electrode had sensitivity 57 mV/dec21. Another sensor based on graphene-coated glassy carbon electrode had sensitivity 59.2 mV/dec and was stable during 3 weeks64. Sensitivity of K+ ISE based on fullerene-modified glassy carbon electrode was 55 mV/dec65.
Calibration of pH ISE (see
The obtained results appear to approve upon existing solid-state pH sensors. For example, pH ISE based on Pt electrode with PEDOT polymer had initial sensitivity of −46 mV/pH, which decreased by 55% after 28 days of storage66. Another pH ISE based on Pt electrode with poly(aniline) had sensitivity of 55-59 mV/pH (depending on ionophore) and remained stable for 1 month67. pH ISE based on graphite rod demonstrated sensitivity −55 mV/pH and was stable during 2 month68. Graphene-based pH ISE had sensitivity −56 mV/pH, but its storage stability was not tested21.
To evaluate performance of the sensors in conditions close to real applications we analyzed artificial urine, prepared as described in Materials and Methods. Five samples of artificial wine with different pH and K+ content were used; the pH and K+ concentrations were chosen from their typical values in urine (reference range for K+ concentration in urine is 25-125 mmol/day, and typical pH range is 4.5-8.069). The results are presented in the Table 2 (below). The difference between the spiked potassium concentration and detected by K+ ISE was 13% or less. The pH ISE detected higher levels of pH than a commercial pH meter; difference was 0.3-0.4 pH values. This shift is probably explained by influence of high ionic content of artificial urine on the sensor's work. However, for medical purposes the accuracy of pH detection can be considered sufficient, since the urine pH is highly variable and in case of diseases it can be shifted to 1 pH value or even more. Furthermore, accuracy of the pH and K+ detection probably will be better in less complex samples or in diluted urine.
Amperometric sensing of the herbicide atrazine on LIG platform was demonstrated. Graphene sensors perform well in terms of enzymatic-based sensing due do the direct electron communication between the electrode and active center of the enzyme.33 Horseradish peroxidase was selected for this enzymatic biosensor due to its high stability and availability70. Our procedures involve the initial detection of hydrogen peroxide. HRP has shown selective measurements of hydrogen peroxide through amperometry71. HRP is a heme group enzyme where the Fe(III) of this group acts as the electron source34. Peroxidases are shown to decompose hydrogen peroxide (H2O2) into water and oxygen34. HRP has been used similarly in detecting the herbicide glyphosate at low concentrations37. Therefore, it is reasonable to use HRP in detecting our analyte in PBS pH 7.4.
The atrazine calibration curve in
The HER electrode was prepared by electroless deposition of nanoscale amount of Pt onto the LIG. The OER electrode was prepared by thermally decomposit the nickel (II) acetate tetrahydrate by photonic heating. The electrocatalytic activities of LIG-Pt and LAG-NiO toward HER and OER in 1M KOH were measured. Linear-sweep voltammograms (LSV) was performed for OER from 0 V to −1.7 V at a 5 mV/s scan rate, and for OER, from 0 V to 1.7 V at 5 mV/s, respectively. The catalytic activity of the LIG-Pt and LIG-NiO are plotted in
Supporting Information for Water Splitting
With particular reference to
Preparation of Water Splitting Electrodes
Preparation of LIG-NiO electrodes. Once LIG electrodes were lasered, 15 of 1M nickel (II) acetate tetrahydrate was deposited onto each electrode. The solution was allowed to dry for 10 minutes. The CO2 laser, running the same etching pattern, lasered the LIG a second cycle to induce the thermal decomposition of the nickel salt into nickel (II) oxide and other gases, such as oxygenates, hydrogen with hydrocarbons, and carbon oxides. The silver paste was applied to the stem of the LIG electrode. Acrylic polish was applied to the electrode to ensure a constant, working surface area of 25 mm2 throughout experiments.
Preparation of LIG-Pt electrodes. The deposition of Pt on LIG is followed by the recipe reported previously (49-52). A chloroplatinic/formic acid solution with a 100:5 ratio, respectively, was used to electrolessly deposit Pt onto bare LIG electrodes. Bare electrodes were placed into a glass vial containing 10 mL of the acid solution. The pH of the solution was changed from 1.68 to 1.75 by adding Ammonium Hydroxide in order to increase the rate of Pt deposition. The electrodes sat in the solution for 24 hours under ambient conditions. In a similar manner to the LIG-NO electrode, silver paste and acrylic polish were applied. See, e.g.,
In conclusion, we report the fabrication of electrochemical ion sensors, pesticide biosensors, and water splitting electrodes by utilizing laser-induced graphene (LIG) platform. The ISE sensors demonstrated wide linear ranges (0.3-150 mM K+ and pH 5-8), near-Nernstian sensitivity (53.0 mV/dec for K+ ISE and −56.6 mV/pH for pH ISE), and good dry-storage stability (100% of the initial sensitivity after 40 days for K+ ISE and 97% after 44 days for pH ISE). These analytical characteristics of the sensors are comparable or exceed previous reports for nanomaterial-based K+ 64 and pH66,68 solid-state sensors. Practical applicability of the proposed ISEs was confirmed by the analysis of artificial urine that contained 11 metabolites and inorganic ions. The characteristics of the sensors make them suitable for various biological applications, such as detection of K+ and pH levels in blood plasma73,74, urine75, and other biological fluids76,77.
Simple and efficient LIG electrodes were developed for potentiometric sensing and water splitting. The HRP pesticide biosensors created by functionalizing the LIG electrodes permitted the selective monitoring of the herbicide atrazine with a high sensitivity (28.9 mA/μM) with negligible inference from other commons herbicides (glyphosate, dicamba, and 2,4-Dichlorophenoxyacetic acid). The LIG electrodes also exhibit small overpotential, 35 mV for hydrogen evolution reaction and 448 my for oxygen evolution reaction at 10 mA/cm2 after platinum and nickel oxide are immobilized. LIG can be used as electrodes directly after lasing, without necessity in any further treatments that are required in case of printed graphene electrodes53. The fabrication procedure is easily scalable for mass production of the electrodes. Such low cost, fast and simple one-step procedure for the electrode preparation also eliminates the need in modification of the electrode with nanomaterials as was necessary in earlier works21,64,66. In summary, the presented LIG electrodes can serve as a versatile platform for the creation of solid-state ISEs, enzymatic electrochemical sensing, and water splitting applications. Additionally, laser induction of polyamide as a fast process could fabricate electrodes on a large scale. The solution phase deposition of metal precursor could also be integrated into roll-to-roll production of such electrodes, which is amenable to potential scale-up and commercialization.
With particular reference to
Growing food demand requires increasing agricultural yield, which is ensured largely in part by the application of pesticides. However, the over application and migration of pesticides can affect vital insect populations and harm the environment. Current methods to measure pesticide levels and prevent over applications are time consuming, complex, and not economical. As such, there is a need to develop a time-efficient, economical sensor for the accurate detection of pesticides. One specific group of pesticides, neonicotinoids, has been shown to be specifically toxic toward honey bees and environmental biodiversity. As such, four different kinds of neonicotinoids were analyzed specifically in this work: clothianidin, imidacloprid, thiamethoxam, and dinotefuran. A laser induced graphene (LIG) device was developed and used in a standard three electrode electrochemical cell setup. The sensor showed a linear range of 10-40 μM and detection limit of 823, 384, 338, and 682 nM for clothianidin, imidacloprid, thiamethoxam, and dinotefuran, respectively. These results indicate that LIG sensors can be utilized for the selective detection of neonicotinoids without the use of a biorecognition agent.
The development of neonicotinoid insecticides over three decades ago is considered a major success in agrochemical research due to their ability to eliminate a broad spectrum of sucking and certain chewing pests that can damage croplands, home yards and gardens, and golf course greens.1 Neonicotinoids exhibit physicochemical advantages over previous generations of pesticides for pest management that have led them to become the fastest growing insecticide used; neonicotinoids account for over 25% of the global pesticide market.2 Neonicotinoids act within target pests by interfering with the acetylcholine neurotransmitter ultimately causing neurotoxicity and they operate systemically by dispersing throughout a plant system which consequently causes the plant to become toxic to specific herbivores.3 Hence, neonicotinoids have negatively impacted a large number of non-target organisms including a variety of aquatic species4 and honeybees5 primarily due to the significant down-regulation of gene expression related to metabolism and detoxification7 More specifically, imidacloprid (DAD), thiamethoxam (TMX), clothianidin (CLT), and dinotefuran (DNT) (including their variants S-dinotefuran and R-dinotefuran) are the most widely used neonicotinoids and they have been shown to be toxic to non-target species such as honeybees with acute oral LD50 ranging from (0.004-0.005) μg per bee.9 8, 10 Hence these four major neonicotinoids have started to diminish the biodiversity of the ecosystems in which they are used.6
The role of neonicotinoids causing human disease (e.g., cancer) and their long-term effects on ecosystems cannot be conclusively pinpointed due to poor neonicotinoid exposure assessment.22.22 Neonicotinoids are becoming more prevalent in a variety of ecosystems especially in rural agricultural settings as neonicotinoids present in high concentrations in seed dressings can drift to neighboring areas during the seed sowing process and only 1.6-20% of the active neonicotinoid ingredient in the seed dressing is absorbed by the plant, leaving the majority in the soil. Hence these agrochemicals have percolated into watersheds and even drinking waters in the agricultural regions of the Midwestern United States (i.e., in the state of Wisconsin) including in private portable wells at high concentrations (TMX: 1.43 μg/L, IMD: 1.59 μg/L, CLT: 3.88 μg/L). Perhaps more concerning is a recent study that found neonicotinoids in treated municipal drinking water in the state of Iowa (another state of the Midwestern United States) which suggests that conventional water treatment practices are unable to eliminate neonicotinoids from the water supply. Hence wide-scale knowledge and mapping of neonicotinoid drift, runoff, exposure, and endpoints is critically important information to help oversee appropriate neonicotinoid stewardship and application methodologies in order to minimize their environmental impact.
Currently, there are no commercially available in-field sensors available for wide-scale neonicotinoid testing. The standard method for quantifying concentrations of pesticides from field samples requires packaging the contaminated samples of soil or water and shipping them back to a laboratory for analysis using gas or liquid chromatography coupled with mass spectrometry.25 These methods are accurate and reproducible for identifying and quantifying analytes, but are time-consuming and expensive. They require extensive sample preparation, highly trained personnel and specialized instrumentation. Inherently, these methods cannot provide real-time or in-field analyte concentrations nor be used to screen large sample sets in a timely and cost-effective manner. Commercially available pesticide field sensors are few and generally consist of colorimetric test strip papers (e.g., Pesticide Detection Test Cards by RenekaBio, Agri-Screen®, Tickets by Neogen, and OrganaDx by MyDx) that are limited to qualitative (high/negative) outputs, limited to testing organophosphates and carbamates, and easily fouled/misread due to particulate matter found in fieldsamples.30
Electrochemical-based biosensors offer a promising solution to monitoring neonicotinoid concentrations in a rapid, quantifiable, low-cost manner that is suitable for large-scale use. Electrochemical biosensors are widely researched for in-field sensing as they are not affected by optically dense, turbid fluid that can negatively affect colorimetric sensors and they provide an electrical signal that can be directly correlated to analyte concentration levels.32 Various reports of electrochemical neonicotinoid sensors exist in the literature using a variety of biorecognition agents including molecular imprinted polymers, aptamers, antibodies, quenchbodies, and (Q-bodies), with favorable results for monitoring TMX, IMD, DNT, and/or CLT down to detection limits of indicated in those publications. The limitation of these sensors of course is that they only can be used once (antibody/aptamer binding to target molecules generally cannot be undone within the context of in-field test conditions), suffer from limited shelf-life due to biorecognition agent degradation, have been limited to monitoring just one or more neonicotinoids. To create biorecognition element-free neonicotinoid sensors, researchers have used a variety of materials including bismuth, β-cyclodextrin (β-CD), silver, and metal-organic frameworks (MOFs) to directly reduce/oxide the pesticides to create a measurable electrochemical signal. However, the construction of such sensors is tedious requiring sputter coating, hydrothermal nanoparticle synthesis, nanoparticle chemical coprecipitation synthesis, magnetic separation, centrifugation, solvent soaking/rinsing, and results are poor. Some work has even attempted to monitoring all four of these four neonicotinoids. Perhaps some of the more promising results in terms of sensor sensitivity and longevity, have come with sensors based on carbon nanomaterials. A variety of carbon-based nanomaterials have been used for neonicotinoids sensing including graphene oxide, nanoparticle decorated carbon composites, graphene and graphene oxide modified glassy carbon electrode, macro-meso-microporous carbon composite. However, these sensors are not produced in a scalable fashion as they require multiple chemical processing steps including centrifugation, solvent soaks, washing, stirring/mixing, high temperature pyrolysis (800° C.), heated/fuming acid etching, and electrode polishing to create the carbon nanomaterials and to cast them onto electrode surfaces. Moreover these sensors are not able to be used continuously or have low shelf-life due to the use of biorecognition agents, and/or have not shown to be capable of measuring signal from all four major neonicotinoids, viz., TMX, IMD, DNT, CLT.
Perhaps one of the more scalable routes to creating carbon nanomaterial-based electrochemical sensors has been with the use of laser-scribed graphene often referred to as laser induced graphene (LIG).12 LIG is created through a direct-write process that circumvents the need to create graphene/graphene oxide via chemical synthesis (processes that often include high temperature pyrolysis and/or high temperature acid etching as used in the Hummer's or Brodie method) or create graphene through high-temperature chemical vapor. LIG also eliminates the need for chemical synthesis of graphene and the subsequent ink formulation associated with more scalable printed graphene electrode processes (e.g., screen, gravure, inkjet, and aerosol printing) as well as eliminates the need for mask, stencils, and pattern rolls associated with screen and gravure printing for example. Moreover, LIG eliminates the need for post-print annealing (e.g., rapid-pulse, thermal, or photonic annealing) that is needed to evaporate ink solvent and carbonize ink binders (e.g., ethyl and nitro cellulose) to make the printed graphene sufficiently conductive for electronic applications such as sensing. The multi-layered, turbostratic structure of LIG improves the physical properties (more resistant to shear stress) of the graphene as compared to conventional AB-stacked (Bernal) structured graphene. Furthermore, the porous and high-defect nature of LIG improves heterogenous charge transport during electrochemical sensing which consequently improves target analyte to electrode mass transport/binding, reduces the ohmic reduction in electrical potential, and leads to the ability to measure small currents within low sample volumes.15 Hence researchers have used LIG to electrochemically sense a wide variety of target analytes including sensing fertilizer and hydrations ions in soil and sweat respectively20,21, monitoring of biogenic amines and Salmonella bacteria in food samples16,22, impedance-based cell monitoring23, aptamer-based biosensing in serum17, and biomarkers in sweat24. To our knowledge, only one example exists of using LIG for pesticide detection exists in which the enzyme organophosphorus hydrolase was immobilized on the graphene electrode detection of the organophosphate methyl parathion on actual crop surfaces in real time.19 These results are promising but the irreversible inhibition of the enzyme does not permit multiple uses and its shelf-life would be limited to the use of a biorecognition element. There does not exist in the research literature to date a LIG sensor capable of neonicotinoid monitoring.
Herein, we report the first example of using LIG for neonicotinoid sensing. We demonstrate how the biorecognition element-free LIG electrode without the use of biorecognition agents can be used to selectively monitor the four major neonicotinoids (CLT, IMD, TMX, and DNT) with a rapid response time (˜10 s) through the use of square voltammetry. More specifically the sensor is capable of monitoring these major neonicotinoids from other pesticides that are commonly used in conjunction with neonicotinoids in the Midwestern United States including other broad-leaf insecticides such as parathion, paraoxon, and fipronil as well as systemic herbicides such as glyphosate (Roundup®) atrazine, dicamba, and 2,4-dichlorophenoxyacetic acid (2,4-D). The reported sensor obtained low detection limits (CLT—823 nM; IMD—384; TMX—338; and DNT 682 nM) which are below the regulatory threshold level (xx nM) in fresh water.27 The corresponding limits of detection for. Finally, neonicotinoid monitoring was confirmed in actual complex biological matrices by river water samples collected from the South Skunk River in Iowa.
The fabrication of LIG consists of preparing the polyimide (PI) substrate 12, positioning the laser 30, laser rastering which induces the graphene 14, and the application of acrylic polish 17 and silver ink 91 (
The graphene morphology and thickness were characterized using scanning electron microscopy (SEM,
The chemical compositions of the obtained LIG were analyzed by X-Rays photoelectron spectroscopy (XPS) (
i
p=2.69×105AD1/2n3/2v1/2c (1)
The ESA was calculated as 0.313 cm2 which is 160% of the geometric surface area (0.196 cm2).
LIG electrodes 80 made by the above-technique (see
The current reduction peaks increased with increasing concentrations of neonicotinoids (
Next, the electrode response to all four neonicotinoids introduced simultaneously in the same test vial was monitored. To this end an overall neonicotinoid calibration was modeled to predict the concentration of a combined neonicotinoid solution (
Furthermore, the neonicotinoid reduction potentials were compared with potential interference species. These pesticides include the common herbicides atrazine, glyphosate, dicamba, and 2,4-dichlorophenoxyacetic acid as well as common insecticides fipronil, parathion, and paraoxon (
Recovery tests were performed in water from the South Skunk River in Iowa. The water was filtered through a 0.45 μm filter and brought to 0.5 M NaCl. The solution was spiked with 40 μM of a neonicotinoid. For comparison, neonicotinoids were detected in deionized water that was also brought to 0.5 M NaCl. The recovery percentages are presented in Table 4 (below) and were calculated using the river water recording as the found response and the deionized water recording as the standard response.
In conclusion, this work demonstrates one of the first studies to electrochemically determine a family of pesticides, specifically the presence of neonicotinoids, without the use of a biorecognition agent. The reported sensor showcases the first use of LIG as a viable platform for the rapid detection of neonicotinoids and demonstrates a more scalable route to multilayered graphene electrodes than more conventional fabrications methods that use low throughput CVD or chemical/mechanical exfoliation of graphite for graphene synthesis and methods that use screen, gravure, aerosol, inkjet printing to form the electrode. The lack of a biorecognition agent allows for easy use, ease of fabrication, and less stringent storage protocol. Additionally, the detection of neonicotinoids was not affected by other common herbicides and insecticides, thus bolstering the reported platform as highly selective. The sensor accurately detected neonicotinoid concentrations in complex media such as river water, with accuracies of 114.3, 105.0, 160.0, and 92.4% for clothianidin, imidacloprid, thiamethoxam, and dinotefuran, respectively. The sensor showed a linear range of 10-40 μM and detection limit of 823, 384, 338, and 682 nM for clothianidin, imidacloprid, thiamethoxam, and dinotefuran, respectively.
The broader implications of this work could be significant for those working in monitoring or mapping pesticides across entire watersheds/ecosystems as well as for those seeking to improve electrochemical electrode technology in general. The reported sensor circumvents the need to transport river water and soil samples to a lab for chromatographic analysis and allows for on-site testing. Hence this technology could permit rapid testing of agricultural runoffs and river water and help policy makers set guidelines on when and where such pesticides can be used. Furthermore, the creation of LIG electrochemical electrodes could be useful for a wide variety of applications that require high surface area and defect rich graphene for heterogenous charge transport such as with energy storage elements such as supercapacitors.
Phosphate buffer saline 10 mM potassium chloride pH 7.4, clothianidin, imidacloprid, thiamethoxam, dinotefuran, atrazine, glyphosate, 2,4-dichlorophenoxyacetic acid, dicamba, paraoxon, parathion, and fipronil were purchased from Sigma Aldrich. All pesticides were prepared in deionized water (18.4 MΩ-cm) except imidacloprid, clothianidin, and fipronil which were prepared in acetone and 100 times diluted in deionized water. Kapton polyimide film (125 nm) was purchased from DuPont.
A 75 W CO2 Epilog Fusion M2 Laser was used to convert PI into graphene. A 5 mm diameter working electrode was lased using speed 15% (271.9 mm/s), power 7% (5.25 W), 1200 dots per inch, and a defocus length of 2 mm. The contacts were covered with silver paste to mitigate wear on the sensor. Acrylic polish was used as a passivation layer to control the working electrode surface area.
Palmsens4 potentiostat was used for all electrochemical measurements. Square wave voltammetry was performed in a 5 mL volume of PBS pH 7.4. The potential was stepped from −0.6 to −1.5 V with a potential step of 5 mV, amplitude of 25 mV, and frequency of 25 Hz. A three-electrode setup was used: LIG working electrode, commercial Ag/AgCl reference electrode, and commercial platinum wire counter electrode. Square wave voltammetry was run 10 times in bare buffer to purge the buffer and electrode surface of any redox species that was electroactive in the potential window. Sensors were calibrated to each pesticide by then mixing the pesticide for 2 minutes at 300 rpm. Stirring was then turned off and the scan was performed. Sensors were washed 2 times with deionized water before each addition of pesticide. Sensors were disposed of after use. Interference pesticides were tested at 40 μM. Results were reported as a change in current from the initially recorded baseline. The combined neonicotinoid model was calibrated to the average responses of each neonicotinoid for the corresponding concentrations. The accuracy of the model was tested by mixing 10 μM each of thiamethoxam, dinotefuran, clothianidin, and imidacloprid. This served as a model for 40 μM of neonicotinoid solution. Additionally, sensors were tested in a solution of river water brought to 0.5 M NaCl. Percent accuracy was reported for 40 μM concentration spikes compared to the current response found in 0.5 M NaCl deionized water which was used as the standard.
Those of skill in this technical field will appreciate that the foregoing embodiments are not limiting. The invention can take a variety of forms.
One example is that variations to the disclosed embodiments and examples are possible, as would be appreciated and within the skill of those skilled in this technical art.
Other variations are possible. Below are a few:
1. Laser.
Lasers are featured in the above embodiments. The inventors have data to show UV lasers can be used to convert sp2 carbon into sp2 carbon to produce graphene. The quality of the graphene produced will depend on the laser used and the power density (J cm−2) delivered to the film. CO2 lasers have shown to be better than UV to produce LIG.
2. Laser Operating Parameters.
The operating parameters that work for that Laser for the LIG patterns, functionalization, and pathogens in the specific embodiments above can vary. They can vary from the indicated states or settings even though they may not produce optimal results. They can still produce effective results, which is intended to mean they can detect an analyte with sufficient accuracy, repeatability, and precision as to be useful for a given application. This includes, but is not limited to, effectiveness at least on the order of effectiveness of at least some state-of-the-art electrochemical or bio-sensors of the types discussed herein. There is a range of operating parameters that would produce effective results in this sense. It is envisioned that a range of laser power densities (J cm−2) delivered at polyimide film that would be effective to produce LIG-based patterns. This information would be universal for different lasers (e.g., UV and CO2 laser). As a rule, the settings are primarily dependent on the laser type, i.e., for CO2 laser the variables are focal lens offset [mm], laser speed [cm s−1], and laser power [W].
3. Laser Beam and its Optics.
In the specific examples, a lens is typically used with the with the laser. The optics used with the laser can vary according to need or desire. A main requirement for the embodiments is a converging lens. Multiple size lenses can be used, but each lens will have an effect on the required lens offset [mm], laser speed [cm s−1], and laser power [W]. Currently, we are determining the upper and lower limit of the beam diameter. The theoretical limit of LIG line width is the same as the smallest beam diameter possible.
4. LIG.
At least in the exemplary embodiments LIG can be accomplished in one pass of the laser. One pass of the laser can formulate LIG on a polyimide surface. Multiple passes can be used to produce LIG, also when there is a need to change the hydrophobicity of LIG produced with one pass. LIG formation is determined by combination of characterization results. First electrically (e.g., the use of a multimeter and sheet resistance measurements); second, electrochemically (e.g., cyclic voltammetry and electrochemical impedance spectroscopy); third, spectroscopic results (e.g., Raman spectroscopy and X-ray photoelectron spectroscopy).
5. Substrate from which LIG can be Generated.
The primary example of a substrate from which LIG can be produced is polyimide. Other groups have demonstrated that any carbon precursor that can be converted into amorphous carbon can be converted into LIG upon further treatment with a CO2 or UV laser. Some examples include polysulfones, poly(ether imide), and polyphenylene sulfide. In general, some level of temperature resistance is desired along with a prerequisite of a cyclic carbon structure. Lower quality LIG have been shown in dried coffee grind, coconut shell, and dried wood, among others.
6. Functionalizations
Use of electrodes according to the present invention can be implemented in a variety of ways. Non-limiting examples of different functionalizations or applications with which electrodes according to one or more aspects of the invention have been discussed supra. As will be appreciated by those skilled in this technical art, variations on those are, of course possible, according to need or desire.
Background information regarding some of the applications can be found at the following, each of which is incorporated by reference herein:
US20200025753A1 to Claussen et al. discusses the basics of electrode-based immunosensors.
US2019/0088420A1 to Tours, et al. discusses basics of electrode-based water splitting and energy harvesting.
U.S. Pat. No. 10,900,925B2 to Chumbimuni-Torres et al. discusses basics of electrode-based ion selective sensing.
The foregoing are for example and not limitation.
This application claims the benefit of Provisional Application U.S. Ser. No. 63/016,068 filed on Apr. 27, 2020, all of which is herein incorporated by reference in its entirety.
This invention was made with Government support under Grant Nos. CBET1706994 and CBET1756999 awarded by the National Science Foundation and Grant Nos. 2020-67021-31375 and 2021-67021-34457 awarded by the National Institute of Food and Agriculture of the US Department of Agriculture. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63016068 | Apr 2020 | US |
