Paper-based microfluidic devices: Fabrication, detection, and significant applications in various fields

3.1.1 Enzymatic methods

In this type of methods, analyte specific enzyme(s) are selected for conversion of the analyte into compound which is either coloured or liberate compounds that are responsible for the production of the colour. These are usually used for biomolecules or similar analytes that have specific enzymes for metabolism. Common examples of such colorimetric reactions are:

The above reaction uses urease enzyme to convert urea into ammonium carbonate which forms a coloured complex in the presence of phenol and hypochlorite [37].

In the above reaction, glucose oxidase enzymes aerobically hydrolyse glucose to form gluconate and H₂O_2. The H₂O₂ so formed reacts with ABTS to form a coloured complex [38].

3.1.2 Non-enzymatic methods

These methods usually involve complexation principle to form coloured complexes with the analytes of interest. But since these compounds are not as specific as enzymes, this method becomes less specific and reliable than enzymatic ones. Common examples include [38,39]:

The colour thus produced is then detected using a simple colorimeter. But in recent practices, instead of colour, intensity of the colour produced is used as a parameter. This gives much more reliable and efficient data. Also, with increasing concentration of the analyte, the intensity of the colour is largely affected, thus in the analytical detection of biomolecules, colorimetry can be used effectively.

4.1.1 Detection of creatinine in urine

Creatinine is one of the most important biomarkers present in the body. Determination of creatinine in urine helps in determining kidney functions efficiently. Creatinine is formed in the body as an end product of creatine metabolism. The reason behind using creatinine as biomarker for kidney function determination is that it is excreted from body mostly through the glomerular filtration at constant rate. When there is any abnormality in the pattern of creatinine excretion such as increased or decreased levels, impairment of kidney functions can be detected. Additionally, concentration of creatinine present in urine also works as an index for urine dilution in body. Originally, creatinine assays were done in laboratories using Jaffe’s reaction, but it lacks specificity. So, Talalak et al., in 2015, [54] proposed a low-cost enzymatic paper-based method for detection of creatinine in urine. This method involved generation of H₂O₂ because of enzymatic action of creatininase, creatinase, and sarcosine oxidase on creatinine present in urine sample. H₂O₂ hence produced reacts with 4-aminophenazone and HTIB giving pink red quinoneimine dye which was detected colorimetrically. This method showed linearity range between 2.5 and 25 mg·dL⁻¹ of creatinine (r ² = 0.983), and limit of detection (LOD) was found to be 2.0 mg·dL⁻¹. Although, this method is 10–20 times less sensitive than conventional methods, but unlike conventional methods, sophisticated instruments, trained analysts, tedious sampling techniques etc., are not required and it also offers portability and time and cost effectiveness. This proposed method thus provides us with low-cost detection method for creatinine to be used for low-income and developing countries.

4.1.2 Detection of thiocyanate in saliva

Thiocyanate is an endogenous molecule present in the physiological fluids due to consumption of thiocyanate-containing foods such as milk, cheese, glucosinolates containing vegetables, etc. But when the human body is exposed to tobacco smoke, the thiocyanate ion levels in the body are increased abruptly because of the hydrocyanic acid present in the smoke of tobacco. The reason behind this is production of thiocyanate as a detoxification of cyanide ion in the body is carried out by the reaction of cyanide with thiosulphates in the liver. Thiocyanate ion concentration in body can be determined from any physiological fluid but in cases of smoking, these levels are pronounced in saliva. Various reported methods for determination of thiocyanate ion in the body involved UV-Vis spectrophotometry, fluorimetry, atomic absorption spectroscopy (AAS), surface-enhanced Raman Spectroscopy (SERS) etc., but all these methods were time consuming and non-economic, producing large amounts of waste products with consumption of high volume of solvents. Thus, Pena-Pereira et al., in 2016, [18] devised a paper-based analytical method for detection of thiocyanate ion in human saliva. This study proposed a paper-based device of about credit card size with 40 test zones on which detection was carried out with Fe(iii) reagent solution as a pre-loaded agent. The matrix used in the study was Whatman No. 1 filter paper. The detection reaction involves colour formation by production of iron(iii)–thiocyanate complex which is red coloured in nature. The detection was done using HP-4500 desktop scanner/printer for recording of images and using blue channel for recording intensities. Channel blue was selected after carefully comparing results in grayscale, red, green, and blue channels. The proposed study shows LOD of 0.06 mM for thiocyanate with RSD of 3%. Recovery values in recovery studies were found to be in the range of 96.1–103.6%. These results were compared to reported UV spectrophotometric method and differences were found to be not significant analytically. Thus, this method provides analytically reliable results as compared to previously reported methods and at the same time proves to be a time and cost-effective method by consuming very few solvents and producing reproducible and reliable results in noticeably short period.

4.1.3 Detection of IL-5, HBsAg, and IgG as biomarkers

Interleukin (IL)-5, HBsAg, and IgG are important biomarkers in our body which are indicative of various diseases. IL-5 is primarily produced in CD4⁺ T cells and functions to regulate growth, differentiation, activation, and survival of eosinophils in the body. Its increased levels in the body act as a critical biomarker for bronchial asthma. Similarly, HBsAg is an important qualitative biomarker for hepatitis B virus infection in the body which is the major reason behind hepatic damage and hepatocarcinoma worldwide. IgG acts as biomarker for detection of Neuromyelitis optica, an inflammatory condition characterised by demyelination of optic nerve and spinal cord. Originally, various highly sophisticated methods were used for determination of these biomarkers such as heterogenous immunoassays, chemiluminescence, thermal lens microscopy, etc. Although these methods were very much sensitive and selective, but being highly sophisticated and tedious, these methods proved to be expensive and non-portable. In 2009, Yu et al. [55] proposed a dextran-based PDMS-fabricated microfluidic device for the determination of these biomarkers. This device was fabricated using soft lithographic techniques and allowed on-line real time detection of biomarkers in ELISA. This proposed method used a series of reaction involving dextran, PDMS, sodium periodate, hydrochloric acid, and hydrogen peroxide to produce luminescent complex that was detected colorimetrically. The study shows LOD in the range of nanograms of analyte and hence proves to be sufficiently sensitive method. The levels of IL-5 were found to be 90.04, 98.67, and 94.34 ng·mL⁻¹ when spiked with 100 ng·mL⁻¹ of standard. Similar reproducible results were also observed for HBsAg and IgG. This proved the proposed method to be more time efficient, greener, and portable in which detection devices cost was almost negligible as compared to the traditional instrumental methods.

4.1.4 Detection of glucose in blood as biomarker of diabetes

Diabetes is one of the major causes behind deaths among all the diseases. It is the most common cause of blindness, renal failure, strokes, heart attacks, limb amputations, etc. According to WHO, diabetes cost 1.6 million lives directly in 2016. Such dangerous disease should be detected at as early stages as possible. One of the most important biomarkers for detection of diabetes is blood glucose levels. Normal fasting blood glucose level is 100 mg·dL⁻¹ and within 2 h of eating, normal glucose levels are up to 140 mg·dL⁻¹. If glucose levels rise above normal, then the person might be suffering from diabetes. One of the easiest diagnostic parameters of diabetes is blood glucose level, for which Ornatska et al., in 2011, [56] proposed an instrument free, paper-based analytical device based upon the principle of colorimetry for detection of blood glucose levels. This study devised a method involving microfluidic device coated with ceria nanoparticles that change colour in the presence of H₂O₂ evolved out in the reaction between glucose from serum and glucose oxidase enzyme pre-loaded on the paper device. The device was first optimised using various concentrations of H₂O₂ and then glucose and their calibration curves were prepared. The r ² value for calibration curves were found to be 0.965 and 0.985 for H₂O₂ and glucose, respectively. For recovery studies, 100 mM glucose was added and recovered values were calculated to be 102.2 (±4.4) mM for 11 runs with RSD of 4.3%. For analytical purposes, linearity curve of glucose was constructed between 0 and 35 mM concentration (r ² = 0.963) and glucose content was determined to be 3.71 mM in non-processed, no additives human serum sample (normal level = 3.6–5.8 mM glucose). Thus, this method proved to be sensitive and efficient when analysing blood glucose levels. Also, in the study, reusability of nano-ceria coated test strips was checked, and strips were found to be re-usable for at least ten repeated assays.

4.1.5 Detection of G6PD in blood

Haemolytic anaemia is a quite common disorder characterised by destruction of red blood corpuscles at a rate higher than their rate of generation. A most common phenomenon that can contribute to haemolytic anaemia is deficiency of glucose-6-phosphate-dehydrogenase or G6PD enzyme. Although G6PD deficiency is a genetic disorder, it is usually triggered when any drug causing oxidative stress to cells is administered. Under such conditions of oxidative stress, excessive haemolysis starts to occur in the body of G6PD deficit person causing haemolytic anaemia which can also prove fatal if use of drug is not stopped or anaemia is not controlled. For such patients, checking G6PD levels becomes necessary before administration of any drug that can cause stress to cells. For this purpose, various testing methods such as biochemical G6PD activity test, methaemoglobin reduction test, cytochemical test, fluorescent spot test, etc., are available but these tests are very costly and require sophisticated instruments, trained personnel, and are not portable. So, in 2016, Kaewarsa et al. [10] proposed a method of G6PD detection based on paper-based analytical devices which is a lab-on-chip kind of device that can be used as portable, solvent friendly, cost effective, and time economic method for prevention of acute haemolytic crisis. In this study, the group fabricated a device on Whatman Filter paper No. 4, by using wax printing technique. The fabricated device was pre-loaded with reagents like Glucose-6-phosphate, NADP⁺, and TNBT which produced red coloured formazan complex detected by using desktop scanner and recording intensities from the colour observed. The calibration curve for varying concentrations of G6PD was prepared and r ² value of 0.973 was observed. 151 samples of blood were tested for G6PD levels and results were compared to the gold standard biochemical G6PD activity test and precision was found to be 5.38% and 11.91% for G6PD normal and G6PD deficit samples, respectively. Although the proposed method is not as much sensitive as compared to the gold standard test, but it gives reliable results in relation to G6PD levels and can be used to detect G6PD levels at the spot in minimal time.

4.1.6 Detection of dopamine in blood

All the functions in the human body are controlled by different neurotransmitters such as acetylcholine, adrenaline, dopamine, etc. Among these, dopamine has very important role in the nervous system in functions of behaviour, mood, circadian rhythm, cognitions etc., of a person. Normal concentrations of dopamine are in nanomolar to micromolar ranges (from 10 nmol·L⁻¹ to 1 µmol·L⁻¹) of extracellular fluids of body. Even minute changes in this concentration of dopamine can result in various psychological disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and Parkinson’s disease. Conventionally, levels of dopamine in body have been determined using various methods such as enzymatic assays, chromatographic techniques, and capillary electrophoresis. These methods are extremely sensitive but at the same time require high precision, costly solvents, trained professionals, and tedious instrumentation for testing. To overcome this, Liu et al. in 2018 [27] reported a paper-based analytical device-based method for easy, portable, time economic, and cost-effective detection of dopamine in blood samples. The device was fabricated on Whatman Filter paper No. 1 using solid ink printer with consequent heating for effective formation of distinct hydrophobic and hydrophilic zones. The fabricated device was loaded with ferric chloride and phenanthroline solutions. On addition of dopamine, ferric ions are converted into ferrous form which reacts with phenanthroline giving red coloured complex. This red coloured complex is detected using iPhone 5 camera and the intensities were recorded using Adobe Photoshop software. The recorded intensities were processed as a function of concentration of dopamine in the sample. The study shows a detection limit of 0.37 µmol·L⁻¹ of dopamine with r ² value = 0.999 for the calibration curve of the same. The LOD of the analyte in this study was found to be similar to that of an already reported study performed using a highly sophisticated instrument, hence proving a sensitive and effective method. The recovery using the proposed method was found to be 99.4–99.6%. Thus, the proposed study helps with convenient, disposable, and time economic alternate of traditional analytical methods.

4.1.7 Detection of H₂O₂ and glucose using starch-iodide-gelatin µPAD

Glucose is one of the most important metabolic products in the body. It also acts as biomarker for diseases affecting glucose metabolism and in some cancers such as Islet cell tumours. Thus, detection of glucose with high sensitivity, selectivity, and efficiency is one of the prime needs in biochemical detection methods. Microfluidic paper-based analytical devices have emerged as important technique for such detections using relatively simpler solvents, being very time and cost effective and highly portable methods. In a study by Liu et al., in 2019 [57], a starch-iodide-gelatin-based microfluidic paper device was proposed. This study used a device fabricated using starch, potassium iodide, and gelatin mixture. H₂O₂ was detected using starch, potassium iodide, and gelatin, whereas glucose was detected using starch, potassium iodide, gelatin, and glucose oxidase system. Analytes were tested in concentration ranges of 0.5–6 mM for H₂O₂ and 0.5–5 mM for glucose, and their LOD was found to be 0.1 and 0.05 mM, respectively. For real time studies, real human serum samples were tested for Glucose and recovery values were found to be in the range 95.7–97%.

4.1.8 Detection of lactoferrin in human body fluid

Lactoferrin is one of the glycoproteins present in our body and it is also known as biomarker for various diseases. Various traditional or conventional methods are present which are based on immunoassay and show selectivity and high sensitivity but is not suitable for point-of-care testing. Kudo et al. [58] in 2019, developed a colorimetric detection method using µPADs. In this method, the color intensity can be judged by naked eye and also using smartphone for colorimetric sensing for quantitative lactoferrin analysis due to high affinity of lactoferrin to ferric ions. Lactoferrin concentration is estimated according to colour change in complex-encapsulated particles formed by displacement of indicator from a calorimetric by lactoferrin. This method shows the LOD to be 110 µL·mL⁻¹. This proposed method can be used for routine analysis of lactoferrin in blood.

4.1.9 Detection of SARS-CoV-2 humanised antibody

SARS-CoV-2 is a 30 kb single-stranded positive-sense RNA that is more than 89% identical to SARS-CoV [59]. The symptoms of infected people range from mild flu-like symptoms to severe bilateral pneumonia and death [60]. Thus, diagnosis of COVID-19 infected patients is urgently needed to execute the quarantine procedure to isolate them from the rest of the population, limiting the spread of the virus. A timely and accurate testing method is required for the diagnosis of SARS-CoV-2 infection. The World Health Organisation (WHO) currently recommends reverse transcription polymerase chain reaction (RT-PCR) as one of the most accurate and sensitive methods for determining SARS-CoV-2 infection because it directly detects viral RNAs in the sample [61]. However, collecting the sample and performing this diagnosis techniques necessitate highly trained medical personnel and advanced instruments, as well it is an expensive procedure. A relatively quick, sensitive, and low-cost platform could aid in alleviating the aforementioned challenges, particularly in underdeveloped nations. Kasetsirikul et al. [62] created a paper-based ELISA for the detection of SARS-CoV-2 humanized antibodies. Because of its low cost, ease of use, and small sample volume, paper-based ELISA is promising for diagnostic applications. The paper device was coated with recombinant SARS-CoV-2 nucleocapsid antigen and laminated for easy sample handling. In the presence of 3,3′,5,5′-tetramethylbenzidine substrate, this assay used a colorimetric reaction followed by a horseradish peroxidase (HRP) conjugated detecting antibody (TMB). By using digital image processing, the colorimetric readout allows for naked eye inspection and quantifiable data. According to the results, this assay can detect SARS-CoV-2 humanized antibody at 9 ng·L⁻¹ (0.122 IU·mL⁻¹) with a percent RSD of less than 10%. In contrast, the LOD and repeatability of commercial ELISA kits on the market are 5 IU·mL⁻¹ with a percent RSD of 15–20%.

4.1.10 Detection of biomarkers of bladder cancer

Bladder cancer is a tumour of the urinary system that is more common in men and has a high incidence of recurrence [63]. In clinics, cystoscopy is the gold standard for detecting and tracking bladder cancer recurrence [64]. This method, however, is inconvenient and less sensitive to early detection. Other diagnostics, such as fluorescent in situ hybridisation (FISH) [65], quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) [66], circulating tumour cells (CTCs) [67], and ImmunoCyt, however, require expensive apparatus and skilled operators, limiting their utility. Jiang et al. [68] have conducted a study employing a microfluidic paper-based analytical device fabricated by wax printing to detect bladder cancer biomarkers named nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from urine samples. The detection is screened by ELISA using a pair of antibodies that can combine with NMP22 and BTA antibodies, with phosphate-buffered saline as the control. An antibody from the pair is fixed at the detection line, while the other is coupled to the colloidal gold, based on the idea of an antigen-antibody immune response (creation of a double-antibody sandwich structure). As experiment and control groups, urine samples were taken from 11 bladder cancer patients and 10 healthy people. Out of which only one is a false negative, with eight being double-positive and two being only positive for NMP22. For the urine samples from the ten healthy subjects, no colour was seen in the detection line. These findings suggested that the PAD can be used to detect bladder cancer in urine samples successfully. As a result, the data revealed an astonishing checkout efficiency of up to 90.91%. Meanwhile, this technology allows for home-based self-detection from urine samples in less than 10 min for the entire procedure, providing a new tool for tumour diagnosis, prognosis evaluation, and drug response evaluation that is quick, inexpensive, and simple. The significant results of applications of µPADs devices in bio-fluid analysis have been compiled in Table 1.

4.2.1 Detection of pesticides in vegetables

With the rapid expansion of population, food demands of the economy have risen a lot due to which use of chemical agents in agricultural practices has seen an abrupt increase. With the increased use, chances of presence of pesticides as contaminants in foods and vegetables have thus been raised multifold. Pesticides are usually chemical toxins responsible for improved growth, better disease prevention, and great pest resistance, but at the same time, are more dangerous for human health when consuming foods with pesticide contaminations. Among all the pesticides, organophosphates (OP) are the most common class which are effectively used in agriculture. Thus, better, sensitive, and effective analytical tools are required for their detection in food products. There have been many conventional methods used for the detection of OPs in food products such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS)/MS, enzyme linked immunosorbent assay (ELISA), etc., but all these techniques have their own merits and demerits. A relatively simple and cheap but reliable method is proposed by Nouanthavong et al. in 2016 [69] based on nanoceria-coated paper-based analytical device. The principle behind detection of pesticides in this study was using acetylcholine esterase and choline oxidase activity reduction and sensing using evolved H₂O₂ for colour change in ceria particles pre-loaded on the paper. For analytical purposes, two main OP pesticides methyl-paraoxon and chlorpyrifos-oxon were tested and their detection limits were found to be 18 and 5.3 ng·mL⁻¹, respectively. For actual samples, spiked cabbage (vegetable) and dried green mussel (food) were tested. Recovery values of tested OPs in these tests were found to be ∼95% which were very similar to those of LC-MS/MS analysis, thereby giving reliable and sensitive results.

4.2.2 Detection of chlorpyrifos by competitive inhibition

Use of pesticides have increased rapidly over the last few years to meet food demands in the expanding population. To detect such pesticides present as contaminants in the food products have thus become utmost important in respect to human health. For this purpose, Kim et al., in 2018 [70], developed a paper-based analytical device based on principal competitive inhibition for detection of chlorpyrifos. The study used indoxyl acetate and acetylcholine esterase as pre-loaded agents to be loaded on the fabricated paper. Here indoxyl acetate converts into blue coloured indigo dye if there is any inhibitor of Acetylcholinesterase (AChE) present. When pesticide-contaminated sample is added, pesticide inhibits activity of AChE hence producing colour in indoxyl acetate. In this study, the colour thus produced is recorded by imaging using Canon Powershot G15 digital camera and intensities are recorded using image manipulation software. The detection limit for chlorpyrifos was found to be between 8.6 and 12.46 ppm. The r ² value for calibration curve was found to be 0.96.

4.2.3 Bioactive paper sensor for organophosphates and carbamates detection

Food contamination due to pesticides require rigorous and effective control on limits with safe, efficacious, reliable, and reproducible methods with extreme sensitivity. Organophosphates and carbamates are among the most toxic chemical agents to be used as pesticides with severe fatal and sometimes lethal outcomes. Badawy and El-Aswad in 2014 [71], developed a paper-based analytical biosensor device for detection of organophosphate and carbamate pesticides. Profenofos and methomyl were the main pesticides used as analyte in this study among the organophosphate and carbamate classes, respectively. Biosensor paper was fabricated by spreading the reagents on paper using a painting brush. The spread of reagents included mixture of chitosan immobilised with acetylcholine esterase by glutaraldehyde in the presence of DTNB. When the analyte containing sample was introduced on the paper, the pesticides in the sample caused a reduction in the hydrolytic activity of AChE on acetylthiocholine as a substrate. The yellow colour produced was noted for its intensity and recorded for analytical purposes. Detection limits for profenofos and methomyl were found to be 0.27 mM and 6.16 × 10⁻⁴ mM, respectively.

4.2.4 Detection of nitrite and nitrate in food samples

Sodium and potassium salts of nitrite are used in various food products as preservatives and also to increase the colour and flavour in various vegetables. As intake of these causes health concern, to overcome these Thonkam et al., in 2020 [72], have proposed a method for simultaneous estimation of nitrate and nitrite in food products. In this method they used a µPAD, which uses beeswax as a new hydrophobic material. Beeswax emulsion was applied onto the filter paper with the help of screen printing to form a hydrophobic barrier and thus defining hydrophilic areas in the paper as it permeates in a portion of the paper and it measures simultaneously the nitrite and nitrate using colorimetric detection based on Griess method. Then, the results are measured by measuring the colour intensity using ImageJ of the resultant colour. Nitrite can be detected by derivatising with sulphanilamide and N-(1-naphthyl)ethylenediamine, it is detected on the left side of the paper and in case of nitrate it must be first reduced to nitrite by vanadium(iii) salt and then Griess reaction can proceed, after measuring the colour intensity by using ImageJ and after comparing the two values, the concentration of nitrite and nitrate can be measured. This method has good reproducibility, efficient resolution, and does not require organic solvent or complicated instrument, and the results obtained by the µPAD was closely related with those obtained from the spectrophotometric method. The detection limit of nitrite and nitrate was found to be 0.1 and 0.4 mg·L⁻¹, respectively, and RSD of nitrite and nitrate was found to be 1.8–2.9% (inter-day) and 2.1–3.1% (intra-day), respectively. The proposed method can be used for routine analysis of nitrate and nitrite in food product.

4.2.5 Detection of norfloxacin residues in food

With the growth of large-scale animal agriculture has come a marketable increase in the farmers’ use of veterinary drugs or veterinary medicinal products to prevent illness, treat illness, improve feed efficiency, and augment animal growth [73]. This increase in antibiotic use has unfortunately resulted in an increase in antibiotic-resistant strains of bacteria, a decrease in overall fitness of the animals, and a serious public health risk because these residues frequently pass through the food supply chain and end up in our diets [74]. Antibiotic use on a regular basis can result in unexpected bioaccumulation of these antibiotics in our bodies. Antibiotic resistance, allergenic response, carcinogenicity, mutagenicity, teratogenicity, and disruption of normal intestinal gut flora can all result from such accumulation to a certain level [75]. The fluoroquinolone class is one of the most commonly used antibiotic families in animal agriculture, distinguished by its broad spectrum of action. Norfloxacin, also known as 1-ethyl-6-fluoro-4-oxo7-piperazin-1-yl-1H-quinoline-3-carboxylic acid, is a common member of this family used in animal agriculture. It works by inhibiting bacterial DNA replication. It is a fluorinated third-generation quinolone antibiotic with fluorination at position 6. The complex food matrix, which might impair sensitivity and so necessitates lengthy sample pre-treatment steps, is one of the challenges in detecting antibiotics in meat products. Antibiotic monitoring systems such as spectrofluorometry, HPLC-MS, and ELISA that have been used in the past are labour and resource intensive, necessitate highly trained personnel, are costly, and take a long time. A method for detecting norfloxacin in four different food matrices has been proposed by Trofimchuk et al. [76] using the coffee-ring effect with paper-based microfluidic analytical devices. The sensitivity of norfloxacin determination was improved in this study. In this study, µPADs were created by printing wax channels onto cellulose-based filter paper and then heating the paper to allow the wax to enter the paper. The colorimetric reaction between norfloxacin and the added iron(iii) nitrate nonahydrate in 5 mM ammonia in each reaction chamber was monitored to determine norfloxacin content in the food samples. A noticeable colour shift was caused by the metal ion–antibiotic combination. Following the colorimetric reaction, pictures were captured and analysed using ImageJ software to estimate the relative pixel intensity, which was then used to calculate norfloxacin concentration. The analytical sensitivity of this device was determined to be as low as 50 ppm when studying the inner-ring reaction and as low as 5 ppm while analysing the outer coffee ring, providing for a cheaper, faster, and more user-friendly technique to detect norfloxacin than current methods.

4.2.6 Detection of pesticides residue in food using organic solvents

Assays of AChE inhibition have received a lot of attention as a good way to quickly screen pesticide residues in food safety assessment [77]. The detection is typically predicated on the basis of toxicity mechanism that organophosphate and carbamate pesticides from samples block the activity of AChE selectively, suppressing the AChE catalysed probe molecule hydrolysis [78]. Most organophosphate and carbamate insecticides are well recognised for their low water solubility. As a result, aqueous solution extraction efficiency of pesticide residues in food samples is typically low, resulting in poor responsiveness and incorrect findings. However, most organic solvents, despite their greater extraction capabilities, would poison AChE, and therefore, affect detection accuracy if they were present in the enzyme reaction system [79,80]. Jin et al. [78] proposed a new protocol incorporating AChE inhibition assay with organic-solvent-based sample extraction for the rapid and sensitive detection of organophosphates (phoxim, chlorpyrifos, triazophos, and methamidophos) and carbamates (carbaryl and carbofuran) pesticides in food, which was developed on microfluidic paper-based devices fabricated using wax printing technique. Instead of being pre-immobilised onto the chip substrate, AChE was applied after the sampling and solvent evaporation steps. As a result, enzyme toxicity from sample pre-treatment solvents was totally avoided. From this study it was found that the pesticides chlorpyrifos, phoxim, carbaryl, triazophos, carbofuran, and methamidophos had detection limits of 0.77, 0.39, 0.25, 1.29, 0.006, and 1.39 mg·L⁻¹, respectively.

4.2.7 Detection of S. aureus and E. coli and their antibiotic resistant strains in milk

Milk is an important part of the human diet and is included in many nations’ official dietary standards. It contains nutrients that are difficult to come by in dairy-free diets, including amino acids, vitamins, and minerals [81]. However, milk is not only healthy for people and animals, but it is also a favourable development medium for a variety of bacteria [82]. Escherichia coli and Staphylococcus aureus are important milk-borne pathogenic bacteria that cause a variety of illnesses in people and animals, including food poisoning and gastroenteritis. One of the main factors for the development of antibiotic resistance bacterial strains is the frequent and extended use of antibiotics in feedstock animals. The ability to design effective preventive and treatment plans requires the identification of infections and their antibiotic sensitivity. Asif et al. [83] have conducted research in which a variety of chromogenic substrates were used to test microfluidic paper-based devices fabricated by wax printing for presumptive identification of E. coli and S. aureus and their antibiotic-resistant strains in milk samples. E. coli and S. aureus are detected using chromogenic substrates such as CPRG and BCIP, respectively. Traditional and extended lactam resistant bacteria were identified using the chromogenic substrates nitrocefin and HMRZ-86, respectively. At E. coli concentration of ≥3.6 × 10⁶ cfu·mL⁻¹, significant change in colour from yellow to red-violet when reacted to CPRG was observed. In case of S. aureus, an identifiable change in colour from colourless to mauve/purple was noticed at concentration exceeding 3.6 × 10⁶ cfu·mL⁻¹. The change in colour at a concentration ≥1.3 × 10⁶ cfu·mL⁻¹ confirmed the presence of β-lactam-resistant strains of S. aureus. According to the study, the paper devices had a sensitivity of 90% and selectivity of 100% on 640 milk samples. The important outcomes of analysis of food stuffs through these techniques are summarised in Table 2.

4.3.1 Determination of reactive phosphates in water samples

Water is one of the most important aspects for sustenance of all life forms on the planet. While 71% of the Earth is covered with water, the amount of potable water is very less as compared to the total volume because almost 97% of water is held in ocean bodies. Amidst all this, water pollution is a major reason behind the crisis of potable water availability. Thus, a check on such reasons is a very crucial issue in the present times. For the same, various methods and tests are available including pH tests, clarity tests, biological oxygen demand (BOD) value, as well as sophisticated instrumental methods like HPLC/UPLC etc., are also available. But these require time and expertise for a reproducible and reliable testing. In 2012, Jayawardane et al. [84] devised a paper-based analytical method for detection of reactive phosphate species involving colorimetric detection of analytes based on the formation of phospho-molybdenum blue due to phosphate ion species in water. Technique employed was wax printed fabricated device for analytical purposes. The reported device was characterised in the working concentration range of 0.2–10 mg·L⁻¹ of phosphorus with LOD and LOQ of 0.05 and 0.16 mg·L⁻¹ of P, respectively. The observed results were reproducible and reliable as compared to the standard testing methods with extremely low requirement of solvents, equipment, and expert professionals.

4.3.2 Tetrazine-based nitrite determination

Nitrite is an especially important component in chemistry playing an imperative role in many synthetic pathways in addition to physiological, food, and environmental functions. Conventionally, nitrite analysis was done in many ways such as spectrophotometry, potentiometry, amperometry, and luminescence, but all these techniques had few demerits like controlling pH and temperatures or using carcinogenic agents. To overcome these, Ortiz-Gomez et al., in 2016 [85], reported a microfluidic paper-based analytical device involving detection and quantitation of nitrite based on formation of dye on reaction with tetrazines. The study employs acidic medium to convert nitrite into nitrous acid responsible for reaction with s-dihydrotetrazine producing pink colour. With optimisation of parameters like pH, sample volume, and reaction time, the detection limit for this method was found to be 1.30 µM. The method was found to be highly reproducible and reliable and was also applied for detection of nitrite ions in water and compared to standard procedures.

4.3.3 Determination of water hardness, total phenol, and pH

Water is one of the most important resources in our life, therefore its quality control is very important, there are many problem regarding the quality of water like water hardness, acidity, and the presence of toxic organic species. The dissolved calcium and magnesium ions in water define the total water hardness, water hardness less than 60 mg·L⁻¹ is soft, 61–120 mg·L⁻¹ level is considered moderate hard, 121–180 mg·L⁻¹ is considered hard, and above 180 mg·L⁻¹ is considered very hard. The determination of water hardness is carried out by complexometric titration with EDTA. Da Silva et al., in 2020 [86], proposed a device based on colorimetric measurements using a smartphone camera and a µPAD. In this method, the samples are added on the µPAD containing the indicator and then the images are captured by smartphone and lastly the treatment based on the generated histogram. The hardness is determined by reaction between EBT and Mg²⁺ and Ca²⁺ and the formation of complex between them and is compered using the colour contrast. The pH determination is observed by change in colour between the protonated structures. The method found the linearity to be in range of 20–560 mg·L⁻¹ for Ca²⁺ and Mg²⁺ (hardness), 0.20–16 mg·L⁻¹ for catechol, and pH from 4.7 to 12, and limits of detection of 0.083 mg·L⁻¹, 0.124 mg·L⁻¹, and 0.262 for water hardness, catechol, and pH, respectively. This proposed method can be used for routine analysis of water quality.

4.3.4 Detection of phosphate in water

Water is one of the most important things in our life. Groundwater is contaminated in various ways and high concentration of contaminants like phosphates can impact our health. So, to address this problem, various methods are proposed like microelectrode integrated with microfluidics system, but this method is too complicated, large quantities of samples are required, and was also difficult to monitor. So Waghwani et al., in 2019 [87], proposed a method which uses µPAD for quantification of phosphates in water by colorimetric assay and RGB value of colour produced quantifies the phosphate concentration. It detects the phosphates in water by the use of reagent stored in paper. The µPAD is printed with the help of rubber stamp PDMS material and kept in the oven to parch and form the hydrophobic barrier and then the chemical are applied on the paper. The test is carried out by applying a single drop of water and results are obtained. The results obtained by ImageJ is specific and more precise. This method is very excellent because it uses low volume of fluid, has high specificity, and low cost. The concentration range of 120 mg·mL⁻¹ of phosphate can be detected for which only 1 mL of water is required. This proposed method can be used for routine analysis of water.

4.3.5 Detection of bacterial sample in water

Microfluidic paper-based device is used in various applications and to analyse complicated biological samples. Lin et al., in 2019 [88], proposed a method for fabrication of µPADs with water-based polyurethane acrylate. In this method, the filter paper is coated with water-based polyurethane acrylate and then covered by a mask with designed patterns. It is then exposed to UV lamp and then rinsed with water. Water-based polyurethane acrylate is used as it can withstand surfactant solution and organic solvent. This method is used as it are useful for analysing complicated biological samples. Assay of E. coli was done by dropping the bacteria lysate onto the colorimetric substrate of the hollowed circles and after some time the colour intensity was measured by seeing the colour change from yellow to red-violet. The LOD was found to be 3.7 × 10³ cfu·mL⁻¹ and linear response was found to be in the range of 10⁴ to 10⁹ cfu·mL⁻¹, R ² was found to be 0.065 and at varied concentrations of 10⁵, 10⁶, 10⁷, and 10⁸ cfu·mL⁻¹, the RSD of colour intensity were found to be 3.15%, 3.62%, 2.26%, and 2.78%, respectively. This method can be used for routine analysis of bacterial detection like E. coli and other bacterial samples in water.

4.3.6 Detection of nitrate in water

Nitrate is an ion that occurs naturally as part of the nitrogen cycle [89]. It is also an important ingredient for plant growth, but when present in high amounts, it contributes significantly to water nutrient pollution [90]. Nitrate in water comes from a variety of sources, including fertilisers or manure runoff, air deposition, agricultural sources, septic tanks, and wastewater treatment plants. Because nitrate is the most stable form of nitrogen in oxygenated conditions, all other nitrogen-containing molecules in water can likewise serve as a source of dissolved nitrate [91]. Drinking nitrate-rich water raises the risk of colorectal cancer, thyroid disorders, and birth abnormalities in the central nervous system. There are numerous methods for measuring nitrate levels in water that are currently in use. These traditional procedures, on the other hand, necessitate costly instruments and time-consuming analysis. They also necessitate special sample handling and preparation, which necessitate the use of qualified staff. Charbaji et al. [90] has developed a microfluidic paper-based device, fabricated via wax printing, to detect nitrate in water. It was a simple paper-based design with folding architecture. The detection zone’s quality and uniformity, as well as the detection limit, were increased by folding the detection zone over the reagent pad. In water, the proposed nitrate device has detection and quantification limits of 0.53 and 1.18 ppm, respectively.

4.3.7 Determination of pesticides in water

Pesticide use in agriculture has increased, posing a risk of pesticide accumulation in soil, agrofood products, and water, resulting in pollution that is hazardous to living things and human health [92]. Pesticide residues, even at low levels, can have a significant impact on live animals’ neurological systems if they enter the food chain [93]. Furthermore, pesticide residues are accumulated in rivers and ponds as a result of rain or irrigation runoff near areas where pesticides are widely used, resulting in water pollution. As a result, a simple, portable, and easy-to-use analytical device for determining pesticides is still necessary. Fernández-Ramos et al. [94] proposed a study presenting a bioactive microfluidic paper device, fabricated using laser cutting technique, for the determination of organophosphorus and carbamate pesticides in water, using chlorpyrifos and carbaryl as models, respectively. Enzyme immobilised original and effective paper design as the sensor platform is employed for detection. The signal transduction is caused by the inhibited enzyme hydrolysing the substrate, which causes local pH changes in the sol–gel entrapped indicator, as well as a colour change, which is measured with a photographic camera using colour coordinates as the analytical parameter for pesticide quantification. The suggested approach was used to successfully determine carbaryl and chlorpyrifos with a LOD of 0.24 and 2.00 g·L⁻¹, and a repeatability of 4.2% and 5.5%, respectively.

4.3.8 Detection of ammonia in industrial wastewater

Ammonia (NH₃) is a chemical that is widely used and produced in a variety of industries. In wastewater, it is the most prevalent nitrogenous contaminant [95]. Because of its toxicity, the presence of ammonia in wastewater is a major concern. Ammonia toxicity can be harmful to fish and other aquatic animals, resulting in dissolved oxygen depletion and eutrophication [96]. It has already been documented that aquatic species can die at concentrations as low as 0.2 ppm (mg·L⁻¹). Nxumalo et al. [96] has proposed a method for colorimetric identification and quantification of ammonia in industrial wastewater using µPAD. The device was fabricated by wax printing. This study has produced a low-cost monitoring system for ammonia detection in wastewater, a modified version of the colorimetric approach employing Nessler reagent integrated with microfluidic technologies. The method can detect ammonia in the range of 0–5 ppm and has a detection limit of 3.34 ppm.

4.3.9 Detection of bisphenol A (BPA) in water

US Environmental Protection Agency (EPA) defined endocrine-disrupting compound as “an exogenous agent that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental process” [97]. Compounds such as dioxin and dioxin-like compounds, polychlorinated biphenyls, pesticides, and plasticisers are examples of endocrine-disrupting substances, which are classified as emerging pollutants because they can disrupt human hormonal functions [98]. BPA, an endocrine-disrupting compound, which is used in the industrial manufacturing of epoxy resins, polycarbonate plastics, and lacquer coating, to mention a few, has drawn significant attention from the scientific community due to its toxicity and presence in the environment [99]. Several studies have linked BPA exposure to diabetes, heart disease, obesity, breast and prostate cancer, sperm quality problems, neurotoxicity, and polycystic ovarian syndrome [100]. For BPA quantification, variety of analytical techniques are available, for instance, HPLC, ELISA, gas chromatography-mass spectrometry, and molecular imprinted sensor. However, the abovementioned techniques are time consuming and require costly instruments. The rapid and sensitive detection technique of BPA is thus an important issue in analytical chemistry for quickly assessing the safety of water and food samples, as well as the pollution of surface waterways. Jemmeli et al. [98] have proposed a highly sensitive paper-based electrochemical sensor for an enzymatic-free detection of BPA method. For a reagent-free analytical tool, this sensor includes the full electrochemical cell printed on filter paper as well as the reagents for the measurement loaded in the cellulose fibre network. The working electrode was printed with carbon black-modified ink, and the paper was fabricated by wax printing technique. The combination of low-cost nanomaterial carbon black with cheap filter paper enables the construction of a low-cost, high-sensitivity sensor for BPA measurement at the microgram level in river and drinking water samples. The results of water analysis in different samples are depicted in Table 3.