Recent Trends and Advances in Design of Rapid Tests for Colorimetric Detection of Staphylococcus aureus

2.1 Nanoparticles-based colorimetric detection of Staphylococcus aureus

In recent years, gold nanoparticles (Au NPs) have been frequently used in the fields of electronics and optics, especially in bioanalytical and biomedical fields, thanks to their unique properties. Au NPs have more advantageous properties than others like easy synthesis, short production time, stable structure (can be stored at focal temperature for a long time), high surface area, easy attachment of functional groups to the surface and most importantly, unique optical properties. Au NPs are used quite actively in applications in the field of bionanotechnology due to their excellent optical properties. Au NPs have begun to be used as biosensors due to the plasmon resonances that they create in the visible region when they interact with light. When Au NPs interact with an analyte, the presence of the analyte and its concentration in the environment can be measured by the change in the optical properties of Au NPs [10, 23, 24, 25, 26, 27, 28, 29].

S. aureus can cause infections ranging from simple food poisoning to life-threatening invasive infections [30]. It can escape the host immune response and causes these infections by producing biofilms and antiphagocytic capsules, persisting intracellularly, and inhibiting leukocyte chemotaxis. S. aureus may cause skin and soft tissue infections (such as impetigo, folliculitis, furuncle, carbuncle, and cellulitis) as well as necrotizing fasciitis by invading muscle and fascia. It infects bones and joints, causing septic arthritis and osteomyelitis. In the literature, S. aureus is reported as a causative agent of severe necrotizing pneumonia in immunocompromised or alcoholic patients [31, 32, 33, 34, 35]. In addition, MRSA is the main causative agent of hospital-acquired invasive infections. It forms a biofilm that causes bacteraemia associated with infections associated with invasive devices (such as intravenous catheters) and difficult-to-treat infections of artificial heart valve endocarditis [36, 37, 38]. MRSA also leads to surgical site infections, one of the most important complications of surgical procedures [39, 40, 41]. S. aureus also has exotoxins. Food poisoning and toxic shock syndrome are infections caused by staph aureus through its toxins [42].

Penicillinase synthesis is present in more than 95% of staphylococcal strains worldwide; therefore, penicillin is not a treatment option. Penicillinase-resistant penicillins (methicillin, nafcillin, and oxacillin) and quinolones may be used in local infections or if susceptible based on antimicrobial resistance test results [43, 44]. First-generation cephalosporins (such as cefazolin and cephalothin) are also options for surgical prophylaxis, depending on local antimicrobial resistance data. Ceftaroline and ceftabiprole are fifth-generation cephalosporins and are effective against MRSA. Methicillin-resistant bacteria are resistant to all beta-lactams, including cephalosporins [45, 46, 47]. Vancomycin is a glycopeptide and should be the first choice in the presence of MRSA bacteraemia or penicillin allergy in MSSA. Teicoplanin is another glycopeptide that penetrates tissues better than vancomycin [48]. Daptomycin is a cyclic lipopeptide antibiotic and is effective against biofilm. Indicated for MRSA bacteraemia if vancomycin-induced nephrotoxicity is present [49, 50]. Dalbavancin and oritavancin are lipoglycopeptide antibiotics newly approved by the FDA for skin and skin structure infections caused by MRSA [51]. Linezolid is a bacteriostatic antibiotic that can be used to treat MRSA bacteraemia, skin infections and pneumonia where other treatments are contraindicated [52]. In biofilm-associated infections such as endocarditis and osteomyelitis, combination therapy with rifampin or aminoglycosides may be considered [53].

S. aureus is a pyogenic bacteria and is seen with many polymorphonuclear leukocytes in the staining of samples taken from the infection site. It grows rapidly on blood agar, forming cream and gold colonies and causing beta haemolysis. Although the gold standard method for diagnosis is culture, it requires a long time. Rapid tests are needed. MALDI-TOF is a common system used routinely to identify S. aureus from positive blood cultures. Fluorescent-based tests that detect antigens and anti-S. aureus antibodies, tissue rapid PCR detection tests, are rapid tests that are in the development phase. Detection of aureus exotoxins in foods and milk is important for the food industry [54, 55, 56].

Recent advancements in nanotechnology are revolutionizing early and precise diagnostics in healthcare. Biosensors based on NMs integrate critical elements such as bioreceptors, transducers, signal processors, and display mechanisms. These sensors benefit from the extensive surface areas and enhanced conductivity of NMs, which enable effective, robust, and reliable biomolecule detection [57]. For instance, the use of Au and silver (Ag) nanomaterials in colorimetric sensors has shown significant improvements in detecting pathogens like MRSA. These NMs amplify colorimetric signals through mechanisms like aggregation, morphological changes, and surface reactions, offering an expanded surface area for biomolecular probe attachment, which enhances signal strength and overall detection accuracy [58]. Various types of sensors, including electrochemical, fluorescent, and mechanical, have been developed to identify MRSA biomarkers, demonstrating effective performance in clinical environments [59].

Colorimetric sensors based on NMs stand out for their ease of use and visual clarity, allowing results to be interpreted without complex instrumentation. NM-based colorimetric sensors combine traditional and innovative techniques, significantly enhancing detection capabilities. The limitations of conventional colorimetric tests, which often suffer from sensitivity and specificity issues, are mitigated by the use of metallic NPs. These NPs provide large surface areas that improve colour change detection and signal clarity by facilitating the binding of recognition molecules like antibodies or enzymes [60].

The application of NMs for pathogen detection is particularly prominent in colorimetric assays. These sensors utilize the electrostatic aggregation properties of NPs to induce detectable colour changes. Au and Ag NPs are particularly effective in this role, enabling straightforward visual detection [61]. Recent reviews highlight the potential of NP-based colorimetric sensors for detecting S. aureus, emphasizing their heightened sensitivity and specificity. By leveraging the high surface-to-volume ratio and reactivity of metal NPs, these sensors offer rapid and accurate pathogen detection through observable colorimetric shifts [62]. Plasmonic NPs are central to the development of rapid colorimetric sensors for bacterial pathogen detection. These sensors generally rely on colour changes caused by NP aggregation or anti-aggregation effects. However, external variables can sometimes lead to false positives, affecting the reliability and selectivity of the methods [58]. To improve performance, innovative approaches such as anti-aggregation techniques and chemical etching are used to refine the plasmonic properties of the NPs. These advancements enhance the sensors’ ability to detect pathogens like S. aureus swiftly, sensitively, and cost-effectively [57].

Yuan et al. [63] developed an Au NP-based colorimetric sensor for detecting S. aureus using aptamer recognition and tiramine signal amplification (TSA). Biotinylated aptamers specifically bind S. aureus to microtitre plates. Through TSA, a large amount of catalase enzyme binds to the bacteria’s surface, accelerating H₂O₂ consumption and turning the solution blue. In the absence of the target bacterium, the solution turns red. The sensor is highly selective for S. aureus because the aptamers bind specifically to unique surface markers on S. aureus, distinguishing it from other bacteria. This method offers a faster and more sensitive alternative compared to other techniques, with a detection sensitivity of 9 CFU mL⁻¹ for S. aureus.

Shahbazi et al. [59] developed a new method for detecting S. aureus DNA by employing Au NP in a colorimetric assay. This technique offers quick detection, taking only 10–15 minutes (min), with a noticeable colour shift that can be observed both visually and through UV-Vis spectroscopy. The assay’s sensitivity surpassed that of the PCR method, detecting DNA at lower concentrations (8.73 ng/μL compared to 30 ng/μL). The method demonstrated a broad linear detection range, high specificity, and precise accuracy in identifying S. aureus DNA, with Au NPs having a higher aspect ratio proving particularly advantageous. Additionally, this approach proved effective in quantifying target DNA in food and clinical samples, suggesting its potential for on-site detection of S. aureus.

A variety of organic and biological targeting agents have been synthesized to specifically detect bacteria and attached to Au NPs, enabling both visual and spectrometric detection of bacteria. For example, Dr DeGrasse developed specific oligonucleic acids called “aptamers” for the enterotoxin B protein found in S. aureus. This specifically selected DNA sequence was intended to selectively bind to enterotoxin B and thus recognize S. aureus [56]. Dr Chen and co-workers attempted to colorimetrically detect S. aureus by attaching the selected aptamer for enterotoxin B protein to Au NPs [64]. DNA or RNA aptamers have been produced by Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) technology to select aptamers for S. aureus and its methicillin-resistant isolate [65, 66, 67, 68]. Recently, ligands containing boronic acid groups have been extensively used to increase the selectivity and sensitivity in bacterial detection. For example, Dr Wang and co-workers used disulphide phenylboronic acid on Au NPs and used this conjugated structure for colorimetric determination of S. aureus. Disulphide phenylboronic acids on gold nano-spheres bind covalently to cis-diol groups on glycans, forming the wall of S. aureus and stable complexes. However, the biggest disadvantage of the system is that there is only one boronic acid unit in the disulphide phenylboronic acid used and it also weakens the selectivity, sensitivity and binding strength of the system to bacteria [69].

2.2 Catalytic activity-based colorimetric detection of Staphylococcus aureus

S. aureus is a leading cause of both healthcare-associated and community-acquired infections. This bacterium poses a significant health risk due to its virulence factors, including endotoxins, which can lead to severe conditions such as endocarditis, pneumonia, meningitis, toxic shock syndrome, and sepsis. Prompt identification of the infection is crucial for effective treatment. Several diagnostic approaches exist for detecting pathogenic bacteria, including culture methods, biochemical assays, enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR) [70].

All of these techniques are limited by their lengthy processing times, low sensitivity, and the need for intricate sample preparation [71]. Consequently, there is an urgent need for a highly sensitive and rapid approach to detect S. aureus effectively. Recently, there has been extensive research into biosensors. In contrast to conventional techniques, nanotechnology-based biosensors offer enhanced speed, improved efficiency, and greater accuracy [72].

Peroxidase-mimicking activity has gained significant attention for identifying pathogenic bacteria because it offers benefits like cost-effectiveness, ease of use, quick results, and no need for costly instruments. The appearance of a blue colour, resulting from the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂ in a paper-based assay, is easily observable with the naked eye. Hence, there is a pressing demand for developing a novel colorimetric technique for the precise and selective identification of pathogenic microorganisms. Colorimetric biosensors, which rely on observable colour changes, are highly effective for detecting a wide range of targets and biomolecules. Their appeal lies in their visible colour shifts, straightforward operation, and rapid results [73].

Peroxidases represent a broad category of enzymes that facilitate the oxidation-reduction reactions dependent on H₂O₂, thereby mitigating the harmful effects of peroxides and certain aromatic compounds (electron donors). Additionally, these enzymes can drive the transformation of chromogenic substrates into coloured products that can be measured using spectrophotometric techniques. Consequently, peroxidases are extensively utilized in biochemical assays [74]. Hosseini [75] introduced a novel, rapid, and highly sensitive colorimetric approach for detecting S. aureus by utilizing DNA-templated nanoclusters as bioreceptors to identify whole bacterial cells for the first time. The method is highly specific to S. aureus because the aptamers in the nanoclusters bind only to S. aureus, while other bacteria like Salmonella Typhimurium, Escherichia coli, and Pseudomonas aeruginosa do not trigger the same response. This technique was successfully employed for the selective detection of bacterial cells in samples of milk, orange juice, and human serum, ensuring accurate detection even in complex samples.

The present study is a straightforward and dependable colorimetric assay for the sensitive and selective detection of S. aureus, utilizing IgY-Fe₃O₄/Au nanocomposites as capture probes and apt-AuNPs as optical signal enhancers. The assay leverages the enhanced catalytic properties of Au NPs, achieved through etching, combined with a TMB/H₂O₂ reporting system. This approach significantly improves the catalytic performance of the modified Au NPs, leading to a greater signal difference between positive and negative samples. The IgY antibodies and thiol-modified aptamers are specifically designed to target and bind to unique surface proteins of S. aureus. This selective binding inhibits the catalytic activity of the modified Au NPs, ensuring that only the presence of S. aureus leads to the observed colorimetric response, allowing for precise detection even in complex matrices. S. aureus detection is possible with a low limit of detection (LOD) of 10 CFU/mL and a broad linear range extending from 10 to 10⁶ CFU/mL. Yao et al. [76] developed a colorimetric immunoassay for rapid S. aureus detection that incorporates etching-enhanced peroxidase-like activity of Au NPs, utilizing IgY-Fe₃O₄/Au nanocomposites and apt-AuNPs to amplify optical signals. This method facilitates easy visual detection of S. aureus at a minimal LOD of 10 CFU/mL and covers a wide detection range from 10 to 10⁶ CFU/mL as shown in Figure 1.

Zhang et al. [77] developed a single-step colorimetric assay for detecting S. aureus by utilizing the peroxidase-like activity of Apt-Fe₃O₄/Au nanocomposites (NCs), achieving high specificity through the aptamer’s selective binding to a unique S. aureus surface target, with a low detection limit of 10 CFU/mL and a broad linear range from 10 to 10⁶ CFU/mL in just 12 minutes. Gao et al. [78] successfully developed porous nanozymes with an Au core and platinum (Pt) shell (Au@PtNEs) that exhibit high peroxidase-like activity and robustness. They employed a double-recognition colorimetric assay that does not require washing for the rapid and specific detection of S. aureus. This approach achieved a detection limit of 40 CFU/mL and covered a range from 5 x 10⁻¹ to 5 x 10⁻⁵ CFU/mL within 1.5 hours.

Liu et al. [79] engineered a new multimetal hybrid Fe₃O₄-Ag-MnO₂ NC, using trisodium citrate for stabilization. They successfully created spherical Fe₃O₄ NPs that are stable, water-soluble, and decorated with carboxyl groups. The resulting colorimetric technique proved highly effective for detecting S. aureus in various food matrices. Additionally, they developed a smartphone-based colorimetric platform employing an enzyme cascade amplification strategy to identify S. aureus in milk. This system involved a cascade bio-nanozyme setup with glucose oxidase (GOx) immobilized on Fe₃O₄@Ag. The method utilized a cascade reaction initiated by H₂O₂, generated through glucose oxidation by GOx, and subsequent colour generation through the natural peroxidase-like activity of Fe₃O₄@Ag with TMB. S. aureus detection was feasible through visual inspection and smartphone measurement, with the platform achieving a low detection limit of 6.9 CFU/mL in just 50 min and demonstrating commendable specificity and sensitivity.

Duan et al. [80] introduced an innovative nanozyme, PHCS@CuNcPOPTP, which exhibits light-responsive oxidase-like activity through a unique chain-linking approach. This organic-inorganic hybrid nanocomposite was synthesized using a single-step Friedel-Crafts alkylation reaction. The resulting material demonstrated exceptional light-driven catalytic properties due to its distinctive sensitivity to light. PHCS@CuNcPOPTP served as a base for constructing various signal probes, such as PHCS@CuNcPOPTP@Au-Ab2/DNA1, PHCS@CuNcPOPTP@Au-DNA1, and PHCS@CuNcPOPTP@Au-DNA2, which incorporated rabbit anti-S. aureus Rosenbach tropina antibody (bs-4582R, Ab2), DNA1, and DNA2, respectively. By repeatedly hybridizing DNA with these probes, a large quantity of PHCS@CuNcPOPTP-DNA dendrimers was generated, facilitating a chromogenic reaction without needing high H₂O₂ concentrations. This approach led to significant catalytic enhancement and minimized background interference. The resulting detection system demonstrated high sensitivity with a broad linear range (from 10¹ to 10⁸ CFU/mL) and an impressive limit of detection (LOD) of 3.40 CFU/mL.

2.3 Paper-based colorimetric detection of Staphylococcus aureus

Paper is often the preferred substrate in biosensor design. It has many advantages [81, 82, 83]. Paper is easy to access and cost-effective. These properties make it suitable for large-scale production. Due to its lightweight nature, it can be easily transported and stored. The cellulosic structure of paper allows molecules to be carried by capillary action. This permits aptamers, antibodies, and various biomolecules to migrate easily. Therefore, paper as a substrate can be easily modified into a sensor. In addition, paper-based sensors can be easily disposed of without the risk of biohazard contamination from incineration.

Microfluidic devices were first developed in 1979 [84]. They allow measurements to be taken from the sample on a micrometer scale. Multiple functions such as sample introduction, mixing, reaction and signal output can operate in one single unit. The advantages include miniaturization, a smaller sample volume requirement, a lower consumption of materials and a lower cost. It also allows point-of-care (POC)/point-of-use (POU) diagnostics with lab-on-a-chip technology. The development of microfluidic devices has been in the direction of paper-based microfluidics for improved sensitivity, selectivity, and portability. In particular, paper-based microfluidic analytical devices (μPAD) have played an important role in the development of biosensor devices in low-resource settings (e.g. LMICs) since it was first proposed by the Whitesides group in 2007 [85].

As a result of developments, μPADs generate a signal output using a variety of sensing methods such as magnetic, optical, distance, and temperature-based readout. These sensing capabilities allow them to detect microorganisms as whole cells. In addition, pathogen nucleic acids and other biomarkers such as antigens, metabolites and toxins can also be detected using these techniques. Following the publication by the Whitesides group [86], it is now possible to sensitively detect pathogens in a wide range of samples.

Over the past decades, since μPAD was first reported by the Whitesides group in 2007 [85], studies have shown that its potential for mass production and commercialization is very promising. In addition to being cost-effective, accessible and stable, its porous structure allows for easy modification with various chemical and biological reagents. The capillary effect allows the movement of molecules without the need for an external power supply. The white background color allows excellent colorimetric detection and makes the difference visible to the eye [81, 82, 83].

μPAD can provide colourimetric, fluorescent and chemiluminescent signals by optical [86, 87, 88, 89, 90] and electrochemical [91, 92] methods. Sensitivity and selectivity have been enhanced by the use of technologies such as nanotechnology and the CRISPR/Cas system. Thus, there has been an expansion in the areas of application for μPAD. Suea-Ngam et al. [90] developed a colorimetric test for the detection of MRSA that provides rapid, sensitive and quantitative results. The colorimetric test was developed as a silver nanoplate (Ag NPls)-based μPAD. The researchers were able to obtain a qualitative colourimetric result with the naked eye. They were also able to perform a quantitative analysis using a smartphone camera. The test gives results in 30 min and a linear response (R2 = 0.994) was obtained between 1 and 104 copies.

A new generation of NM-based detection methods has been developed with the advance of technologies such as microfluidics and NMs. Paper-based sensors prepared in the form of lateral flow assays (LFA) provide colorimetric results with NMs. In particular, Au NPs and silver nanoparticles (Ag NP) are used. The localized surface plasmon resonance (LSPR) properties of the mentioned metallic NPs are utilized. For example, Lu et al. developed an aptamer-based LFA test for the detection of S. Typhimurium, E. coli O157:H7, and S. aureus. The color change is based on AuNPs. The ability of AuNPs to appear red in colloidal form and purple in aggregated form has been utilized. Colorimetric response can be observed by eye after 10 min incubation [93, 94].

Guo et al. [95] developed a test for the detection of pathogens in infected wounds that can perform rapid sampling, photocontrolled release and SERS detection. The triple-function adhesive tape can detect P. aeruginosa and S. aureus. It can detect bacteria directly from the wound after several hours of incubation. This test shows great promise for use as a POC test device.

Wang et al. [96] studied the simultaneous detection of Acinetobacter baumannii (AB), E.coli (EC) and multidrug-resistant S. aureus (SA). The developed test provides results in 35 min and is a μPAD containing dual aptamers. In the developed test, aptamers were first fixed to the paper layer of the sensor by UV crosslinking and blocking. Secondly, an aptamer-biotin conjugate was conjugated. Finally, HRP-streptavidin and TMB were applied. A blue color change was observed in the presence of bacteria. The LOD was visible to the naked eye even at 10-fold serial dilutions.

Nanoclusters have been used for the detection of S. aureus due to their nanozyme activity. Bagheri Pebdeni et al. synthesized Au/Pt bimetallic nanoclusters using cytosine-rich single-stranded DNA for the detection of S. aureus. Au/Pt bimetallic nanoclusters oxidized TMB in the presence of H₂O₂ due to their peroxidation properties. When TMB is oxidized, specific areas of the sensor change color from clear to dark blue. The catalytic activity of the nanoclusters is reduced in the presence of bacteria. This leads to the production of less oxidized TMB, and consequently, the intensity of the blue color decreases [75]. The working mechanism of this paper-based sensor is shown in Figure 2.

Suaifan et al. developed an assay based on the proteolytic activity of S. aureus. The assay detects proteolytic activity using a magnetic nanobond peptide probe immobilized on a gold platform [97].

A paper-based sensor array capable of detecting sepsis in children was developed by Sheini et al. The working mechanism of the sensor array is based on measuring changes in the optical or electrical properties of the indicator after interaction with bacteria or metabolites released during bacterial growth. Au and Ag metal nanoclusters were immobilized on Whatman paper. The developed sensor was used to detect pathogens in pediatric serum samples. It detects sepsis-causing bacteria such as S. aureus, Streptococcus pyogenes, E. coli, and P. aeruginosa. Changes in fluorescence emission intensity in the presence of the pathogen were recorded in less than 15 seconds using a UV lamp and a smartphone. The researchers quantitatively analyzed the color changes using ImageJ software. The detection limit was 80 CFU/mL and a linear calibration curve was obtained in the range 10²–10⁸ CFU/mL. This test enables the diagnosis of bacteria in human serum and the detection of S. aureus in food samples such as milk, orange juice, and human serum [98]. The colorimetric changes of this sensor array in the presence of bacteria are shown in Figure 3.

In a study by Sekar et al. in 2024, 6 different pathogens were detected using glucose-functionalized gold nanoparticles. Phyto-synthesized AuNPs (Glu-AuNPs) act as transducers. The color intensity of the paper-based colorimetric sensor increased with bacterial concentration and was measured with Adobe Photoshop software. This sensor is suitable for measurement of real samples [99].

Another study in the same year developed a novel paper biosensor based on a polydiacetylene (PDA) polymer functionalized with fibrinogen (Figure 4) [100]. The fluorophore property is released by proteins that bind to fibrinogen on the surface of S. aureus. The binding causes the PDA to bend, resulting in changes in color and fluorescence. The detection limit was 50.1 CFU/mL for S. aureus-contaminated food samples and 65.0 CFU/mL for the pure S. aureus culture. Mechanism of the working principle is shown in Figure 4.

In one of the recent studies, direct loop-mediated isothermal amplification (LAMP) and LFA were combined to detect S. aureus. The limit of detection was 10² CFU/ml with no cross-reactivity with other strains. POC technologies can become more widespread thanks to this developed system. It has advantages such as short test time, high sensitivity and user-friendly application [101].