Chapter 1: Colorimetric Paper-based Sensors for Low-cost Clinical Diagnostics

Biomarkers are biomolecules, such as DNA, lipids, proteins, and small molecules like secondary metabolites, that are expressed differently in disease and normal conditions (Figure 1.1A). These biomolecules perform various functions in the human body, such as storing and transmitting genetic information, catalytic activity, biological regulation, or transporting molecules. They can vary and may be present in physiological fluids, such as blood, saliva, or urine.^1–3  Different biomarkers have been detected and investigated and are now in clinical use. The detection of biomarkers could be performed using standard methods in clinical diagnosis, i.e., polymerase chain reaction (PCR) and immunoassays [e.g., enzyme-linked immunosorbent assay (ELISA)]. These gold standard methods are highly specific and sensitive for detecting biomarkers.^3–7  Other alternative methods can be performed using simple paper-based analytical devices called paper-based devices (PADs).

In this context, the determination of biomarkers is crucial for early diagnosis and prognosis of diseases, including degenerative diseases, and for facilitating effective treatments to enhance health and quality of life. To some extent, they can be useful for monitoring the health status of patients.^1–3,8,9  However, the detection of biomarkers can be challenging, as they are often present at trace levels, particularly in the early stages of a disease,^3,9  and relying on a single biomarker may not provide an accurate diagnosis. Hence, a combination of biomarkers is often required to ensure correct disease diagnosis.^1,2,10  Therefore, these clinical diagnoses often require expensive and complex procedures, including multiple analytical steps, such as washing and incubation, as well as high consumption of samples and reagents. Consequently, these are not widely available in some countries, such as in developing nations, due to low-resource settings or lack of skilled personnel, which may affect precise diagnosis and effective treatment.^3–7  In this regard, gold standard methods, such as PCR and immunoassays, are neither practical nor suitable in developing countries or regions with limited resources.^6,11 

Biosensors are analytical devices or tools that incorporate a bioreceptor as a recognition element, such as enzymes, antibodies or antigens, nucleic acids, or cells, that reacts with and recognizes the target analyte. This reaction is then transduced through optical, electrochemical, mass, or thermal means, converting into a readable signal corresponding to the target analyte.¹  Biosensors can detect a single or multiple biomarkers, with high selectivity and sensitivity, even when they are present at trace levels. In this context, scientists are interested in developing methods based on biomarkers to detect various diseases. These methods could be transformed into point-of-care (POC) devices for use in near-patient settings.^1,3,8,9  Although POC tests are not intended to replace clinical diagnostics, they have recently gained attention, particularly for clinical tests, as they offer a low-cost and rapid option for preliminary clinical screening or testing.^9,12  Currently, various clinical diagnostics are available for the rapid, low-cost, disposable, and non-invasive detection of biomarkers.^1,13 

Paper, an adaptable, cellulose-based material, has garnered interest and is being used as a platform or substrate for developing PADs, including for POC applications. It offers an alternative to the gold standard methods for detecting biomarkers.^2,4,12  Optical and electrochemical detection methods are often used in PADs for detecting single or multiple biomarkers.^6,7  Colorimetry, among optical detection methods, is a suitable detection technique for use in PADs, offering several advantages, such as simplicity, cost-effectiveness, disposability, rapid detection, and the ability to be detected directly by the naked eye.^5,12–14  Furthermore, the expanding use of smartphones, wireless technology, and operating systems allows for real-time and online biomarker analyses.^15,16 

Paper is composed of cellulose, a linear biopolymer containing d-glucose units connected by β-(1,4)-glycosidic bonds.¹⁰  It is a renewable and biodegradable biopolymer produced by plants, trees, and non-pathogenic bacteria.¹⁰  Paper is widely available and inexpensive worldwide.³  It is a lightweight, biodegradable, and eco-friendly biomaterial^5,17  that can be easily disposed of via incineration.¹²  Furthermore, paper can be easily transformed, processed, stored, and transported.^3,13  This polymer has unique physical and physicochemical properties, such as high porosity, biocompatibility, sorption capacity, thermal stability, strength, and hydrophobicity.¹⁰  As it is inexpensive and widely available, paper is an adaptable material commonly used for writing, packaging, and printing. It also serves as an excellent platform for chemical and biological sensing.¹⁷  Due to its high porosity, paper facilitates the immobilization of reagents and bio-reagents and subsequent drying, making it suitable for developing various analytical tools and devices.^4,9,10  Because of the hydrophilic properties and capillary action in paper, pumps are not required for fluid transport, which enables the development of power-free PADs, such as spot tests, dipsticks, lateral-flow assay (LFA), and microfluidic PADs (μPADs).^4,9,12 

PADs are particularly valuable in resource-limited settings, as they meet the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, and Deliverable) criteria proposed by the World Health Organization (WHO), making them promising POC devices.³  Furthermore, PADs can be integrated with portable readouts, such as smartphones, facilitating a simple readout process.¹⁸  They are cost-effective due to the use of inexpensive materials, such as cellulose, cotton, or plastic. Conversely, nanoparticles (NPs) derived from various nanomaterials have also been used in PADs to achieve high selectivity and sensitivity in detecting target analytes.¹⁹  Furthermore, these nanoparticles can be functionalized with bio-receptors to achieve high specificity for the target analyte. PADs are user-friendly and non-invasive, providing easy-to-interpret analytical results, without the need for skilled operators or advanced instruments. They provide rapid results because the properties of paper, such as capillary forces and porosity, accelerate the analysis process. The components of PADs make them both robust and disposable. Additionally, their user-friendliness allows individuals to perform home self-testing without the need to visit a clinical laboratory.^{2–4,6,12,17} 

The first PADs were developed for semi-quantitative glucose analysis in urine samples. Since then, PADs have been widely used across various fields, including basic diagnostic detection (e.g., pH, humidity, and temperature), clinical diagnosis (e.g., immunoassays, urine analysis, and blood tests), food and beverage quality and safety, water and environmental monitoring (e.g., heavy metals, pathogens, and pesticides), and forensics (e.g., explosives and drugs).^3,12 

Various types of paper are used in the development of PADs, and the selection should be performed carefully based on the fabrication method, purposes, and applications (Figure 1.2).⁵  The paper widely used in developing PADs is Whatman^® paper No. 1, which has a high absorption rate, a suitable thickness (180 μm), and an optimal pore size (11 μm). Another type of paper suitable for developing PADs is Whatman^® paper No. 4, which has a large pore size and high retention rate. Additionally, filter paper, with uniform thickness and capillary properties, is also being used in PADs. Nitrocellulose (NC) membranes are also used in developing PADs due to their smooth surfaces and optimal pore size (0.45 μm). They are commonly used in LFAs due to their functional groups, which enable covalent bonding with immobilized biomolecules, as well as their high retention level.^5,6  Bioactive paper (paper functionalized with biomolecules), such as nylon membranes, and ordinary paper (e.g., paper towels and traditional printing paper) are also used in the development of PADs.^5,6,14  Paper composed of bacterial cellulose has also been used due to its flexibility, robustness, and optical transparency.¹⁰  Besides its inherent characteristics, cellulose paper can be easily modified to achieve desired properties. In this context, it can be modified either by mixing it with organic polymers during production or by treating it afterwards, such as by immersing it to modify its surface properties, making it hydrophobic.¹⁰  Given that the hydrophilic properties of paper can be applied for developing sensor devices, hydrophobic barriers are required to direct the flow of sample fluids and reagents into specific channels, preventing mixing outside the sensing area and reducing contamination. Physical and chemical techniques used to develop hydrophobic barriers on PADs are photolithography, screen printing, wax printing, inkjet printing, laser and plasma treatment, and chemical vapour-phase deposition (Figure 1.2).^3,5,20,21  However, various types of PADs have been fabricated and developed to detect a range of target analytes across different fields, such as clinical diagnostics, food quality, and environmental monitoring.^4,5,12,22  In this context, PADs can be classified into (1) spot tests (Figure 1.3A), (2) dipsticks (Figure 1.3B), (3) LFAs (Figure 1.3C), and (4) μPADs (Figure 1.3D).^4,7,13 

Spot tests are the simplest and oldest format of PADs, providing rapid and cost-effective analysis on paper¹²  for various analytical targets. Spot tests were first developed using litmus paper for detecting acid–base reaction^23,24  and metal ions.¹⁵  In the latter case, colorimetric reagents are used as ligands for metal complexes, causing a colour change in the spot test. These devices have been designed and developed for on-site analysis.¹⁵ 

A dipstick or strip test is another simple format of a PAD, where a paper strip is typically immobilized with reagents or bioreagents that change the colour in the presence of the target analyte, producing a qualitative or semiqualitative result.^11,13  The first dipstick format of PADs was designed to quantitatively detect glucose in urine. Developing dipstick formats is quite simple, and their colour change response is easy to analyse and interpret and can be directly observed by the naked eye. However, they have several drawbacks, such as low accuracy, longer analysis time, and focusing only on qualitative analysis.^5,13  For example, dipsticks are pH test strips that are used to measure pH in various sample solutions, and test strips for urine are currently used for simultaneous screening of multiple clinical disorders, such as kidney disease and diabetes.^11,13,17 

A well-known example of LFA is the pregnancy test.⁷  Over the last few decades, LFAs have been developed with a focus on improving their analytical characteristics,⁴  and they are now important devices for determining various biomarkers in clinical samples. In addition, they have been developed for applications in food safety and environmental monitoring.^7,9,13  The principle behind LFA is that the sample solution, which may contain the target analyte, flows horizontally through various pads by capillary force without external pumping and interacts with immobilized reagents or bioreagents of the device.^6,25,26  Typically, LFAs consist of NC strips placed in a plastic carrier (e.g., polyvinyl chloride), consisting of various components, such as a sample pad, a conjugate pad, a test pad or flowing membrane, and an adsorbent pad,^2,4,26  which allow the sample solution to flow through the device. Generally, the sample and adsorbent pads are composed of cellulose paper or glass fibres, whereas the test pad is typically an NC membrane and the conjugate pad is produced from glass fibres.^2,25  In this format, the pads are arranged accordingly to enable the sample solution to flow laterally once it is applied to the sample pad.^25,26  The sample pad is where the sample is applied, enabling the sample containing the target analyte to reach, react with, or bind to the capture reagents.^7,25  In this process, the sample solution flows through the conjugate pad, which contains coloured particles [e.g., metal nanoparticles (MNPs)], bound to receptors (e.g., antibodies or antigens) that capture the target analyte.^25,26  Then, the formed conjugate passes along the strip to the test pad.²⁵ 

In this case, the NC strips can be modified with various reagents or bioreagents to serve as test pads, enabling capillary action to transport the sample fluid along the strip.^4,9  ln this case, the pad is the detection area where biochemical reactions occur in the recognition element when activated via the immobilization of capture molecules, forming the control line and the test line.²⁶  The presence of the target analyte in the sample causes an interaction at the test line, whereas the appearance of a coloured line at the control line indicates that sufficient sample flow has occurred along the strip.²⁵  Finally, an adsorbent pad is positioned at the end of the strip to maintain continuous sample flow through capillary action, absorb excess reagents, and prevent liquid backflow.^7,25,26  The LFA readout involves detecting the presence or absence of coloured lines on the test line, which can be observed with the naked eye or using an appropriate readout.²² 

Generally, two principal basic assay schemes are used for LFA: (1) sandwich (or direct) lateral-flow assay (sLFA) and (2) competitive lateral-flow assay (cLFA). sLFA is more effective for high-molecular-weight analytes and involves two separate recognition processes, which enhance sensitivity and specificity. In this scheme, the target analyte is captured by a primary antibody embedded in the conjugate pad, forming a complex that is subsequently detected by a transducer, typically a colorimetric probe, paired with a secondary antibody impregnated at the test line. The target analyte aggregated at the test line produces a positive signal, with the intensity of the colour increasing in proportion to the concentration of the target analyte in the sample. However, cLFA is more suitable for small molecules, typically involving a single antigenic agent. In addition, the target analyte competes for and blocks the binding sites of the receptors at the test line, preventing them from reacting with the conjugates. The higher the target analyte concentration, the lower the intensity of the coloured line at the test line.^4,11 

With regard to the recognition element, LFAs can be categorized into two types: (1) lateral-flow immunoassays (LFIA), which are the most commonly used and involve labelled antibodies as receptors for the target analyte, with a colorimetric or fluorescent label for detection,²⁵  and (2) aptamer-based LFAs, where aptamers are used for the target analyte recognition.⁷  However, for signal transduction, colloidal gold NPs (AuNPs) are commonly used as colorimetric labels or probes in LFAs due to their unique optical properties and ease of modification for detecting the target analyte.⁴  LFAs offer several advantages, including low cost, user-friendliness, long shelf life, high specificity and sensitivity, small sample volume, multiplex detection capability, and applicability across a range of fields.^4,10  However, LFAs have certain drawbacks that need to be addressed, such as their specificity and sensitivity depend on membrane matrix and receptors. Therefore, it is essential to explore novel matrices to enhance their analytical characteristics.⁴ 

The first μPAD was introduced in 2007 by Whitesides et al., developed using a photolithography technique, and used for colorimetric glucose detection in urine samples.²⁷  Since then, μPADs have been used not only in various clinical diagnostic applications but also in other fields and areas, including environmental monitoring, food quality and safety, and forensic analysis.^13,17,28,29  Unlike other PADs, μPADs control sample flow through a pattern of flow channels, which enables the analysis of multiple analytes.⁹  The flow channels are formed by patterning hydrophobic materials onto hydrophilic paper, developing hydrophilic channels that direct the sample towards the reaction zone.⁶  In two-dimensional (2D) μPADs, flow channels are patterned using physical or chemical hydrophobic barriers through various techniques, such as photolithography, screen printing, inkjet printing, wax printing, and plasma treatment, while three-dimensional (3D) μPADs are designed by folding different layers of patterned paper.^13,17,28,29  The selection of fabrication technique should be based on factors such as the type of substrates, cost, equipment needed, and fabrication time.

Typically, Whatman^® filter paper No. 1 is used in this type of device due to its uniform thickness and capillary properties. In this case, μPADs require small sample volumes (5 to 10 μL) and provide results more rapidly compared to standard laboratory methods. Furthermore, μPADs offer advantages such as miniaturization, portability, disposability, and user-friendliness. They enable multiple detections through various channel patterns and can simultaneously perform both quantitative and semi-quantitative analyses.^28,29  It also enables on-site detection, which is useful for many resource-limited settings.²⁹  Despite their advantages, μPADs may experience low sensitivity. Therefore, various efforts are ongoing to enhance sensitivity, including the use of pre-concentration techniques.^28,29 

The selection of receptors or recognition elements is based on the target analyte to be detected. Antibodies and aptamers are the most commonly used recognition elements in PAD fabrication for detecting various biomarkers. For example, when nucleic acids are the target analyte, oligonucleotides and aptamers are commonly used. Conversely, for detecting proteins, enzymes, and cells, antibodies and aptamers can be used (Figure 1.1C).²  In addition to these commonly used receptors, molecularly imprinted polymers have emerged as alternative receptors, exhibiting high selectivity, rapid response, and cost-effectiveness.³⁰ 

Immobilization of receptors may occur both on the paper surface and within the nanomaterial used as the probe. They can be impregnated onto the paper using physical or chemical methods. These methods ensure that the biomolecules are retained without affecting their biological activity. To immobilize receptors such as antibodies on tags, chemical covalent binding or adsorption techniques are often used. For aptamers, sequences labelled with amine or thiol groups at the 5′ position can be used to bind to nanoparticles. Furthermore, streptavidin–biotin interactions are often used to immobilize them on NC membranes.^2,25 

Currently, paper-based colorimetric sensors are used in diagnosing various diseases, including cancer,¹⁸  neurodegenerative disorders,³¹  infectious diseases,³²  and other chronic diseases.³³  Colorimetric tests assess the presence or absence of the target analyte by evaluating colouration or colour changes. This colour change can be caused by dyes, enzymes, or NPs such as AuNPs.⁵  Colorimetric tests measure the change in absorption or reflection intensity caused by biochemical or chemical reactions between the target analyte and the colouring agents used as probes or tags.

The intensity of light absorbed or reflected typically corresponds to a change in optical characteristics caused by surface plasmon resonance or a structural change.³  It is the most commonly used detection method in PADs because it enables simple, real-time, cost-effective, in situ detection of various molecules.¹⁴  Moreover, the paper provides a clear background with high contrast when colour changes.¹²  In addition, the sample to be analysed using colorimetric PADs can be obtained through invasive sampling methods, such as serum, blood, or synovial fluid, or through non-invasive or minimally invasive sampling methods, such as saliva, tears, sweat, or urine (Figure 1.1A).¹⁴  Despite their advantages, the colorimetric method sometimes lacks selectivity and sensitivity, leading to a heterogeneous response that can lead to user misinterpretation.¹² 

In some cases, various types of NPs and nanostructures have been used as carriers or targeting intermediates for interface detection on PADs. The integration of NPs into PADs improves various sensor performances, such as sensitivity, limit of detection, dynamic working range, specificity, and selectivity against confounding species.¹⁰  Enzymatic conversion or catalysis of chromogenic substrates is a commonly used method in colorimetric detection and involves the interaction between the substrate and its enzyme, forming an enzyme–substrate complex that produces colour.^5,22 

Enzymes, such as peroxidase, are used to catalyse the oxidation of substrates, leading to colour development.³⁴  However, enzymes have certain inherent drawbacks as biomolecules, such as stability issues, sensitivity to environmental conditions, and challenges related to their modification and production. To address these issues, studies have been performed to simulate peroxidase-like activity by developing nanomaterials with catalytic properties.³⁴  It has been observed that certain nanomaterials, such as metal nanoclusters, exhibit enzyme-like activity by catalysing the reaction between 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide, thereby generating a blue colour. This method has been used for detecting cancer cells.²² 

NPs are also used as probes, as they are relatively easy to synthesize in various conformations, form different types of nanomaterials, and can be easily functionalized with active molecules (Figure 1.1C). These nanoparticles include metal NPs, such as gold NPs (AuNPs) or silver NPs (AgNPs), cerium oxide NPs, magnetic NPs (MNPs), paramagnetic particles, and carbon NPs, such as multi-walled carbon nanotubes (MWCNTs) and graphene oxide (GOx), which have a higher surface area and magnetic properties, enabling the detection of the target analyte at trace levels.^10,34  Marker NPs should be selected based on several factors, including colloidal stability, conjugation efficiency with receptors, and low non-specific binding.

Colloidal AuNPs are an intriguing nanomaterial for biological colorimetry and are commonly used as tracers, exhibiting high potential for enhancing the sensitivity of colorimetric biosensors. This is due to their intense colouration caused by their unique physical and chemical features, including optical and plasmonic properties, simple preparation in different shapes and forms, a well-known synthesis protocol, and excellent stability in both solution and dry conditions.^10,25,34  AuNPs are used to bind with secondary antibodies in immunoassays, and their cleavage or aggregation, caused by specific reactions with the target analyte, leads to colour development. AuNP solutions are red due to their localized surface, which can cause a change in their surface properties, leading to their colour change to purple.³⁵ 

The colour produced by the colorimetric sensor can yield qualitative results visible to the naked eye or facilitate the extraction of quantitative data using readout devices, such as spectrometers, smartphones, cameras, or scanners (Figure 1.1D).^2,5,6,13,14  Smartphones are widely used as readouts due to their beneficial features, including high-resolution cameras, easy-to-use operating systems, and wireless connectivity, which enable real-time or online analysis.^15,16  Imaging should be conducted under controlled lighting conditions to avoid affecting the sensitivity and repeatability of the test. White or black boxes with controlled lighting conditions have been proposed for imaging.^3,4  Fluorescent tags or paramagnetic beads are not visible to the naked eye and require specialized readers for quantitative analysis.²⁵  After capturing the image, it is transferred to a computer with dedicated software that calculates parameters, such as red, green, and blue (RGB) values or colour space attributes such as hue, value, and saturation (HSV). Thus, the concentration of the target analyte is calculated and quantified.³⁶  Real-time online scanning and portability are intriguing properties of POC testing, depending on the miniaturization of devices and the advancement of portable analytical tools or devices.³¹ 

In the case of colorimetric sensors, although the equipment required for image processing is small and portable, it can still be heavier and larger compared to the PAD itself. In this case, the naked eye is the most suitable reader for in situ detection.³⁶  As our eyes are highly sensitive to various aspects, such as colour intensity, hue, and light, this can lead to deviations in the understanding of users. In this context, many scientists have proposed novel display formats to enhance image reading accuracy and facilitate device miniaturization, such as distance-, quantity-, time-, and text-based measurements.^36,37  Among the various types of PADs, µPADs are the most suitable for device miniaturization, as they can use smaller channel sizes in microfluidic devices.³⁷ 

Biomarkers, as described earlier, are biological markers that can indicate a specific disease state or other medical conditions and are found in physiological fluids, such as blood, urine, saliva, and sweat. In this regard, some applications of colorimetric paper-based sensors for detecting biomarkers are presented in Table 1.1, which can be classified into three types: (a) ions (e.g., potassium, sodium, and calcium), (b) small molecules (e.g., uric acid and glucose), and (c) macromolecules (nucleic acids and peptides or proteins). The table presents information on the type of PADs, biosensing mechanism, interface, linear range, limit of detection, and type of clinical samples. The table shows that the nanomaterials used in the paper-based sensors exhibit excellent performance due to better immobilization, which enhances the physical and optical characteristics of the biosensing assay.

Lookadoo et al. developed paper-based optode devices (PODs) for detecting K⁺ ions in biological fluids (Figure 1.4).³⁸  It has been found that this prototype of an integrated detection system offers improved features compared to conventional film optodes (optodes are optical sensors that optically detect a specific target analyte using a chemical transducer) and other PADs, particularly in terms of its user-friendliness, cost-effectiveness, and potential for more effective disease management through telemedicine.

Tsai et al. ⁴⁰  proposed a colorimetric paper-based sensor using AuNPs for the diagnosis of tuberculosis (TB). The sensors detected colour changes in AuNPs due to the hybridization of the ss-DNA probe with targeted ds-TB DNA, leading to various degrees of aggregation and changes in the colour of the solution, corresponding to the concentration of the targeted DNA analyte. It can be detected using a smartphone to obtain rapid colorimetric results with minimal reagent consumption. Under optimum conditions, the limit of detection was achieved at 1.95 × 10⁻² ng mL⁻¹ for TB DNA.

Tan et al.³⁹  introduced a new detection device, termed a “tape sensor”, which facilitates the separation of plasma from whole blood and quantification of total bilirubin through colorimetric diazotization. The paper-based tape detection method mitigates uneven colour distribution due to the “coffee stain” effect, thereby enhancing the accuracy of colorimetric measurements on PADs. The extent of haemolysis in the plasma was assessed using the extraction device, indicating no interference with the total bilirubin determination. The accuracy of the tape-based detection method for measuring neonatal blood samples was confirmed through comparison with the hospital pathology tests. Small sample and reagent volumes, low-cost equipment (desktop scanners), rapid detection (<10 minutes), and low production costs (∼$0.6) highlight the usefulness of the device for POC testing in under-resourced environments. The paper-based sensor is inexpensive, rapid, and user-friendly for measuring the total blood bilirubin concentration in the diagnosis of neonatal jaundice.

Tsai et al. developed a PAD for monitoring TB (Figure 1.5).⁴⁰  They demonstrated that the colour of AuNPs changes upon hybridization of the ssDNA probe with the targeted ds-tuberculosis DNA. They tested the detection system under various conditions, including thermal and temporal denaturation at high temperatures for different oligonucleotide probe sequences. Under optimal conditions, the system detected TB DNA with a limit detection of 1.95 × 10⁻² ng mL⁻¹.

Alizadeh et al.²¹  developed a µPAD immunosensor for the detection of carcinoembryonic antigen (CEA). Primary antibodies functionalized with chitosan were mixed with ionic liquids, followed by the immobilization of glutaraldehyde (GA) through cross-linking on the paper surface. The role of ionic liquids is to prevent nonspecific binding. After incubating the sample, the Co₂(OH)₂CO₃-CeO₂ nanocomposite, which was conjugated with a secondary antibody, was introduced into the sensor. Subsequently, the peroxidase-like activity of the nanocomposites leads to the oxidation of TMB in the presence of hydrogen peroxide, resulting in a colour change. Finally, sulfuric acid is added, changing the colour from green to yellow. The colours can be observed with the naked eye, and the quantitative results are obtained by capturing images with a smartphone and analysing them with dedicated software. The colour intensity value correlates with the CEA level, enabling its determination with a dynamic range of 0.002 to 75 ng mL⁻¹ and a limit of detection of 0.51 pg mL⁻¹. This immunosensor was then tested with human serum for detecting CEA, exhibiting high sensitivity comparable to ELISA.

Kitchawengkul et al. ⁴²  successfully developed a paper-based sensor for detecting total cholesterol (TC) in whole blood, using nitrogen-doped carbon dots (N-CD) combined with a 3D µPAD format. This device provides a simple, sensitive, selective, and cost-effective alternative for determining TC in blood samples, similar to traditional colorimetric measurements.

Wirojsaengthong et al.⁴¹  were the first to demonstrate highly sensitive colorimetric PADs for detecting thiocyanate in urine samples. The photovoltaic cocktail solution consisted of a complex that included tridodecylmethylammonium chloride (TDMACl) as the ionophore, 5,10,15,20-tetrakis(4-octyloxyphenyl)porphyrin cobalt(ii) (l) for ion exchange, 2-nitrophenyl octyl ether as a plasticizer, and polyvinyl chloride as a polymer. The paper optode reacts with thiocyanate by increasing the blue value in the RGB index, leading to a distinct colour change from pink to green in the optode. Based on the composite core design, the optimal parameters yielding the highest sensitivities are 4.70 mmol kg⁻¹ TDMACl and 13.75 mmol kg⁻¹ L⁻¹. This sensor is highly sensitive and selective to thiocyanate, compared with other anions, with an activity concentration between 0.001 and 5 mM and a correlation coefficient of 0.9915 for the linear range. The limits of detection for visual and camera-based measurements were calculated to be 50.0 μM and 1.26 μM, respectively. Moreover, the limit of detection calculated from blank standard deviations was 0.65 μM, and the limit of quantitation was 1.87 μM. In addition, this optode has been successfully used to detect thiocyanate in the urine samples of smokers and non-smokers.

He et al.⁵³  developed a µPAD based on a 3D network polymer hydrogel paper to screen total blood glucose. As outlined in the proposed method, the sample zone and detection zone are positioned on the mushroom-shaped analyser. Plasma diffuses into the sensing region as blood enters the inlet portion of the μPAD, and a 3D metallic polymer hydrogel carrier is embedded in the sensing zone. The Cu complex interacts with oxygen and glucose oxidase (GOx), which is retained within the gel both as a bioactive preservative and as the gel carrier. By using the real and controlled whole blood samples with glucose levels ranging from 3 to 200 mM, the glucose levels or values were selected, and the results were found to be consistent with those obtained using the blood glucose meter, demonstrating the potential for real-time sensor applications.

The detection of CEA for the diagnosis of gastric cancer using a colorimetric method was reported by Wang et al.,²⁶  using sandwich immunoassays and wax-printed μPAD. The primary antibody was embedded in Whatman^® filter paper, while the secondary antibody was immobilized with horseradish peroxidase (HRP). Next, the TMB solution served as a colour indicator, generating a blue colour that intensified with the increasing concentration of the target analyte. In this case, a smartphone-based system was used to measure and display the target analyte level on the screen. The method demonstrated a dynamic range from 0.5 to 70 ng mL⁻¹ with a limit of detection of 0.015 ng mL⁻¹.

The p16INK4a is a biomarker for cervical cancer, acting as a cyclin-dependent kinase inhibitor that is overexpressed in human papillomavirus (HPV)-associated precancerous cells.⁵⁰  Detection of cervical cancer was achieved using a visual reading time of 30 minutes. In this case, AuNPs functionalized with anti-p16 INK4a were used as labels and impregnated on NC membranes. The signal amplification method is based on combining the peroxidase activity of the HRP–antibody conjugate with the peroxidase-like activity of AuNPs. In the presence of an immune complex formed between the antibody and the target analyte in the reaction buffer, HRP catalyses the oxidation of TMB, generating a blue spot visible to the naked eye, while a red colour from AuNPs is observed when the antibody reacts with the target analyte. Then, the responses were captured using a mobile phone camera and analysed by ImageJ. In this case, four cervical cancer cell lines were analysed, and the limit of detection was determined based on the lowest cell count that produced a visible positive response. The HeLa, SiHa, and CasKi cell lines exhibited strong positivity, with LODs of 300, 300, and 3000 cells, respectively.

Nie et al. ⁵¹  proposed a simple method to develop μPADs and used them as colorimetric immunoassays combined with gold-enriched signal amplification to detect prostate-specific antigen (PSA) as a target analyte. Here, markers are used to trace patterns on paper surfaces with metal patterns, without requiring skilled personnel or specific equipment. These markers serve as hydrophobic barriers that direct the flow of hydrophilic solutions. A primary antibody is immobilized on the paper surface for target identification. After target binding, AuNPs conjugated with secondary antibodies were introduced, and finally, the gold-enhanced solution was used. PSA levels were measured within the range of 0.5 to 50 μg L⁻¹, with the limit of detection estimated at ≈360.2 ng L⁻¹.

Kuswandi et al. ⁵²  developed cotton cloth-based microfluidic devices (CMDs) using wax printing technology (batik stamp). In this case, small sample volumes were used for reactions in the sensing areas, which were previously impregnated with sensitive reagents. These sensors can detect and measure components in sweat, such as pH, lactic acid, and sodium ions. Colorimetric measurements were performed using a smartphone camera, and colour image measurement was carried out using a free app (Color Grab) for quantitative assessments or visually by the naked eye for semi-qualitative evaluations (Figure 1.6). The CMDs demonstrated excellent linear correlation with the target analyte (i.e., lactic acid was found at 0.994, sodium ion at 0.998, and pH at 0.994). The limit of detection was calculated to be 45.73 μg mL⁻¹ for lactic acid and 56.46 μg mL⁻¹ for sodium ions.

Although clinical diagnosis involves complex methods that are highly sensitive and selective, these are not suitable for conventional diagnosis. As paper is a flexible material with internal characteristics and modifiability, PADs represent a new strategy that offers a suitable alternative to traditional methods and fulfils the requirements of POC devices. Colorimetric PADs have been used to detect various analytes, such as biomarkers, in various biological samples. This chapter explores the use of paper as a platform in sensor development using colorimetric methods for detecting biomarkers as the target analyte.

Recent major advances in PAD development have led to enhanced signal amplification approaches. This has been achieved using novel materials, such as novel NPs, used as markers to enhance sensitivity and provide a distinct colour signal. The integration of PAD with technology, such as smartphones, is expected to improve both qualitative and quantitative signal detection, enabling preliminary disease screening on-site, outside the laboratory, in hospitals, or in remote areas.

Although a significant number of applications exist for colorimetric determination of biomarkers using PADs, specific implementations remain still rare. Despite current advancements in this field, further study is needed to evaluate the disadvantages of paper-based sensors and propose approaches to overcome these issues, thereby improving the performance of paper-based sensors and allowing commercialization. These challenges can include poor analytical characteristics, such as high detection limits, low specificity, poor stability, the need for multiple detections, and subjective interpretations of results.

The authors gratefully thank the DRTPM, Ministry of Education, Culture, Research and Technology, Republic of Indonesia for supporting this work via the Basic Research Grant 2023–2024 (HPD).