Phosphorous (P) plays an important role in biological systems and is crucial for a variety of central tasks (metabolism, genetic information exchange, cell wall integrity, etc.). However, in the wrong form excessive P may cause detrimental environmental effects on aquatic ecosystems [1] and/or agricultural systems [2, 3]. In biological systems, phosphate plays a vital role in regulating primary productivity, making it a limiting nutrient for agricultural production [4]. For this reason, the widespread use of chemical fertilizers is mainstay (particularly after the Green Revolution in the 1960s), increasing the development of P-based fertilizers through extractive mining [5]. Indiscriminate use of phosphate-based fertilizers can increase P load into water bodies, leading to environmental problems such as eutrophication and expediting water deterioration [6]. While P is ubiquitous in natural systems, much of the excess P in aquatic systems is in a form that is not bioavailable to plant uptake. Abundance of P is clearly a problem, but at the same time scarcity of the finite resource phosphorous and lack of viable recovery/reuse techniques have created a paradox [7], making P management among the biggest environmental/agricultural challenges of the 21st century. Furthermore, access to P controls food security and limits geographical access since the largest reservoirs of P are found in a few countries, generating global shortages [8]. As mined P is a finite resource, the expectation is that fertilizer prices will increase over the years, and disruption of supply chains may ensue as P mines are depleted [8]. As a result, there is a pressing need for conservation and management of P, including recovery and reuse. One of the major problems from a monitoring perspective is that there are few reliable field sensors for quantification of P in near real-time. Thus, analytical techniques to measure P are sorely needed, and precise determination of P speciation is vital and important to guarantee precision agriculture and wastewater monitoring, for example.
Analytical methods for detection and characterization of P are classified into five distinct categories: i) chemical techniques (e.g., ion chromatography, inductively coupled plasma mass spectrometry, colorimetry/spectrophotometry, flow injection analysis, chromatography, and gradient thin film detection), ii) biological techniques (e.g., enzymatic assays, protein labeling, electrophoresis), iii) molecular techniques (e.g., Raman scattering, nuclear magnetic resonance), iv) staining techniques (e.g., fluorescence, GFP-activated cell sorting), and v) chemosensors/biosensors (e.g., electrochemical, optical, magnetic transduction methods) [9]. These analytical methods vary widely in terms of efficacy, accessibility, analysis cost, need for sample pre-treatment, and requirement for trained personnel. Furthermore, the ideal analytical method used may change depending on sample matrix (e.g., aqueous or soil sample), P species of interest, and required resolution (e.g., limit of detection and detection). Chemosensors and biosensors provide advantages such as portability (in-field measurement). Optical sensors have been widely explored by Dr. Wolfbeis’ lab [10] for phosphate sensing, providing insightful inputs for sensing phosphates. However, there is a critical need to enhance the sensitivity, selectivity, limit of detection, and reusability, and development cost for electrochemical sensors targeting P [11].
P has a plethora of different chemical/biochemical forms (in fact, there are thousands), making quantification and characterization challenging. More than 80% of these forms are organic P, and only a small fraction of inorganic P is water-soluble. Ortho-P is the most readily bioavailable form of dissolved inorganic P and may take one of four forms depending on pH (PO43−, HPO42−, H2PO4−, H3PO4) [12]. Monitoring ortho-P in environmental, agricultural, and ecological studies is of upmost importance. Most plants uptake the primary form of ortho-P (HPO42−), although the secondary form (H2PO4−) is also bioavailable to roots [13]. To prevent eutrophication, a maximum allowable contaminant level (MCL) for total phosphorous (TP) of 0.05 to 0.1 mg-TP/L in water has been set by the U.S. Environmental Protection Agency (EPA) in some states, but adoption of total phosphorus criteria in the US has been slow even when MCL is established [14]. However, monitoring TP concentrations with high spatiotemporal resolution is a challenge. The most widely used test for estimating TP is the standard EPA spectrophotometric-based (Method 365.5) [15]. This reagent-based test requires the addition of an exogenous reagent containing ammonium molybdate, antimony potassium tartrate and acid (usually ascorbic, sulfuric or hydrochloric). The reaction converts soluble reactive phosphorus (SRP) to an antimony-phosphomolybdate complex known as a Keggin ion [16]. The elegance of the test lies in the specificity toward SRP as well as the intense color change that can be easily detected. Testing procedures rooted in Keggin ion formation are among the most selective tests for P and are central to water quality laboratories. However, not all research questions are relevant to the restrictions of the testing protocol. For example, most tests based on Keggin ion formation require at least 10 min of reaction time and the addition of reagents and strong acids. Reagent-free chemosensors with a high specificity toward at least one form of ortho-P (ideally primary and/or secondary) would enable high spatiotemporal mapping, enabling new research questions that are based on rapid analysis of ortho-P and other forms of labile P in natural systems. Finally, there is a need for dip-and-read systems that can target one or more forms of P in water samples.
Electrochemical phosphate detection has emerged as a viable approach for ortho-P monitoring due to its potential for portability and miniaturization, allowing in-field deployment. Several studies have been reported for ortho-P detection using different recognition schemes, transduction techniques, P species, and reporting a wide range of limit of detection and sensitivity. For example, a Zinc(II)-dipicolylamine-modified gold electrode was applied for testing phosphate anions (H2PO4–), based on redox activity pyridine units complexed with Zinc, with a LOD of 7.5 × 10–16 M [17]. However, acidification is needed to perform measurements at pH 4, which will require the addition of reagents for environmental water analysis. In another example, molybdate(VI) anions were immobilized within a chitosan matrix deposited on a glassy carbon electrode for phosphate anions (mainly H2PO4−, H3PO4 and HPO42−), reporting a sensitivity of 4.4 ± 0.1 µA/µM and a LOD of 0.15 µM [18]. The sensing principle is based on the reduction of molybdate(VI) anions, which requires the addition of reagents to acidify the sample to pH 2. As another example, a cobalt film on glass was formed via physical vapor deposited for ortho-P sensing (H2PO4–) at pH 4 [19], which a limit of detection estimated to be ∼10–7 M. Among the various electrochemical techniques, the most common include cyclic voltammetry (CV), square wave voltammetry (SWV), and DC-potential amperometry (DCPA) [20].
Laser induced graphene (LIG) is one emerging platform that is gaining much interest in chemosensing and biosensing. LIG is a one-step, facile and scalable approach to fabricated 3D porous graphene electrodes by direct writing on commercial polyimide (PI) films under ambient conditions by using a CO2 infrared laser [21]. LIG-based electrochemical sensors are simple and cost-effective, beyond the advantage of LIG being a material with high electrical conductivity [21]. LIG can be functionalized with the material of interest (recognition element), where changes in the electrochemical response of LIG occur due to the chemical interaction between the recognition element and target analyte in solution. In the last decade since the discovery of LIG, this material has been used for targeting a wide variety of sensors, for example: bacteria [22], viruses [23], sugars [20], and ions [24]. This material is simple to fabricate, and numerous portable devices have been developed using custom form factors that meet sampling needs. Recently, a nano copper-decorated LIG chemosensor for organophosphorus pesticide (glyphosate) was developed by Bahamon-Pinzon et al [25]. To date, there are no manuscripts for detection of inorganic phosphate using LIG.
In this paper, we evaluate a new chemosensor material for targeting ortho-P in water samples based on a 2D sorbent structure (graphene oxide PolyDADAMAC, a.k.a. GO-PDDA). The nanocarbon-polymer hybrid sorbent mimics a group of phosphate-binding proteins and is synthesized using grafting techniques [26]. Based on sorption studies, the material is expected to bind ortho-P across a pH range of 5 to 9 via a combination of electrostatic interactions and hydrogen bonds. The suspended GO-PDDA material has demonstrated ability to be regenerated under acidic conditions (manuscript in progress). Here, we fabricate and test LIG electrodes coated with GO-PDDA sorbent. We tested numerous electrochemical techniques and found that electrochemical impedance spectroscopy (EIS) produced the most reliable results and that the LIG-GO-PDDA electrode surface may be regenerated for ortho-P sensing. We also evaluated selectivity towards other anions (chloride, nitrate, and sulfate). This work is the first analysis of GO-PDDA sorbent as a coating material for development of a reagent-free ortho-P chemosensor.