AuNPs can attach multiple diagnostic probe molecules due to their high surface-to-volume ratio (44). They can be used for long-term diagnostics since they are chemically stable and air- and water-resistant (18). The first reported application of AuNPs in TB diagnosis was a colorimetric assay developed by Gupta et al (45). In that study, oligonucleotides of the Mycobacterium tuberculosis RNA polymerase subunit gene sequence were first extracted and then combined with AuNPs and, at a wavelength of 526 nm, the gold nanoprobe solution stayed pink in the presence of the complementary DNA. By contrast, the solution turned purple without the complementary DNA. The assay takes only 15 min per test, with minimal contamination, as it is performed in a separate tube and allows visualization of the results. Follow-up studies showed that this approach detected TB more precisely and sensitively when compared with the automated liquid culture system and semi-nested PCR (46,47).

AuNP aggregation via salt, such as NaCl and MgSO₄, is the most widely used AuNP-based colorimetric method for TB DNA detection. The amount of TB DNA to be detected is inversely proportional to the salt concentration required to cause AuNPs to aggregate (16,27). However, this may lead to false negative signals when the TB DNA content is low, as the salt concentration required to detect AuNP aggregation is very high. Thus, Tripathi et al (32) relied on ethanol-induced AuNP aggregation to detect TB DNA. Ethanol affected the hydrophobic and electrostatic interactions of AuNPs with DNA, generating dipole-dipole interactions that led to AuNP aggregation. As shown in Fig. 2, a 4 μl 100% ethanol addition to the AuNPs-TB DNA complex could cause aggregation. Without TB DNA, AuNP suspensions did not aggregate despite the introduction of 8 μl of 100% ethanol. This method sensitively detects MTB DNA at ~340 fM levels, amplified with a 0.125 ng ml⁻¹ template and produces results in <3 min. Contrary to salt-based AuNP aggregation methods, the researchers claimed their method is sensitive and reliable for early TB identification. This simple, easy-to-use approach does not require AuNP or oligonucleotide modification or expensive equipment, making it favorable in resource-poor settings.

The local electric field enhancement effect induced by surface plasmon resonance (SPR) can enhance the optical activity near the surface of metal nanoparticles, such as surface-enhanced Raman scattering and fluorescence enhancement. The plasma-enhanced effect of AuNPs has several applications in fields such as biomarkers, sensors, photocatalysis and optoelectronics (48,49). Plasma coupling is related to the size, shape, structure and spatial arrangement of NPs (50). Research in this area can help to optimize biosensor structures. Prabowo et al (39) studied the effect of AuNP shapes on plasmonic enhancement for DNA detection. They bound TB's designed single-stranded probe DNA (ssDNA) with gold nano-urchins and nanorods. Then, both mixtures were adsorbent onto a graphene-coated SPR sensor due to the π-π interactions. During the construction of the SPR sensor, annealing the Au layer increased the sensor's graphene coverage and DNA probe load. In experimental plasmonic activity comparison, gold nano-urchins showed the best amplification, detecting DNA hybridization at fM levels. They conclude that gold nano-urchin-assisted DNA detection offers the possibility of early screening for TB using portable sensors.

Due to their affordable cost, simple structure and easy operation, piezoelectric sensors are becoming a TB detection research hotspot (51). A piezoelectric sensor generates electricity from pressure, acceleration and force. Quartz, Rochelle salt and some ceramics generate an electrical charge when subject to mechanical stress, a phenomenon known as the piezoelectric effect (52). Exploiting the special physical and chemical properties of Au at the nanoscale, AuNPs can markedly enhance the performance of piezoelectric sensors for detecting MTB (53). Zhang et al (37) developed a novel piezoelectric sensor based on AuNPs-mediated enzyme-assisted signal amplification for TB diagnosis (Fig. 3A). The biomarker was the 16S rDNA variable region of TB. AuNPs were coupled to the hybridized detecting probe and grown in HAuCl₄ and NADH solutions to transmit electricity between electrode gaps (Fig. 3B). The piezoelectric system detects TB rapidly and sensitively thanks to AuNPs-mediated signal amplification. The process is simple, fast and suited for developing compact portable equipment.

AgNPs, like AuNPs, are chemically stable, electrically conductive and can possess catalytic activity. Their electron transfer efficiency is superior to that of AuNPs, which have more prominent extinction bands (18). Recent advances have seen the use of charge-neutral peptide nucleic acids (PNAs) as hybridization agents in AgNP-based colorimetric DNA assays, enhancing the process by causing nanoparticles to cluster more rapidly in solution without immobilization, thus boosting DNA hybridization effectiveness. Teengam et al (54) developed a colorimetric DNA detection sensor based on PNA-induced AgNP aggregation (Fig. 4A). They designed a detection probe from PNA with a positively charged lysine modification at its C-terminus (acpcPNA), leading to the aggregation of negatively charged AgNPs and a subsequent swift shift in color. This sensor effectively detected TB oligonucleotides, demonstrating a low detection limit of 1.27 nM, showcasing fast, selective and sensitive DNA detection capability.

Conventional methods for producing AgNPs typically involve the use of reducing agents such as sodium citrate, NaBH₄ and hydrazine. While effective in controlling nanoparticle size, these agents pose significant environmental risks (55,56), prompting the pursuit of greener alternatives. Tai et al (40) devised a method to synthesize AgNPs using oil palm lignin, which is rich in phenolic hydroxyl groups and offers an environmentally friendly and cost-efficient solution for AgNP production. These lignin-coated AgNPs were subsequently bonded to laser-etched graphene nanofibers, enabling the direct linkage of single-stranded DNA to form a TB bioelectrode. To assess the performance of the sensor, they analyzed the ability of DNA samples attached to AgNPs to bind to the target DNA by selective hybridization and mismatch assessment. Electrochemical impedance spectroscopy further substantiated the ability of the sensor to detect concentrations as low as 1 fM, achieving a detection limit of 10⁻¹⁵M based on a signal-to-noise ratio (S/N=3:1) with a signal-to-noise ratio of 3:1. The researchers highlighted that this TB detection method is sensitive and ecologically friendly.

MNPs are generally composed of iron, nickel, cobalt and oxides. MNPs feature a high surface-to-volume ratio, excellent dispersibility and strong interactions with biological molecules (57). Gupta et al (42) developed a giant magnetoresistance (GMR) biosensor to detect TB-specific early secreted antigenic target-6 (ESAT-6) protein. This GMR biosensing assay labels monoclonal antibodies against ESAT-6 antigen with MNPs. In the presence of ESAT-6, MNPs bind to the GMR sensor proportionally to protein concentration, altering its electrical resistance. Simulations of the GMR biosensor have shown that it can detect ESAT-6 at pg/ml levels. Cheon et al (58) developed a colorimetric biosensing system to detect MTB 64 protein (MPT64) using nucleic acid aptamer-modified MNPs. The aptamer on the surface of the MNP initially inhibits its catalase activity. Upon binding with MPT64 in the sample, the aptamer releases, thereby restoring the enzyme activity of the MNP. TB can subsequently be detected within 70 min by measuring the enzyme-substrate fluorescence spectra.

Mohd et al (41) developed a portable sandwich-based electrochemical immunoassay device for clinical sputum TB detection (Fig. 4B). They used Fe₃O₄/Au MNPs to capture anti-culture filtrate antigen [CFP10 (10 kDa)-early secreted antigenic target-6 (ESAT6; 6kDa)] antibody, which is more stable than enzyme-conjugated antibodies. Magnetic Fe₃O₄ particles enhance the chemical stability and biocompatibility of Au. Results revealed an excellent correlation in sensitivity (100%) and specificity (91.7%) compared with the gold standard culture method. León-Janampa et al (36) presented a colorimetric sandwich assay incorporating amino-silanized MNPs functionalized with anti-MTB polyclonal antibodies to detect TB in sputum. The biofunctionalized MNPs enhance antigen capture from biological materials, enabling multiple TB antigen detection and decreasing test time compared with traditional ELISA. This method can also evaluate TB markers in early TB cultures, urine and serum.

Graphene is often employed in sensors designed as reduced graphene oxide (rGO), a cost-effective form produced via chemical and hydrothermal reduction of graphene oxide (83). rGO is favored in biosensor design for its high current density, exceptional electrocatalytic properties, extensive surface area, excellent thermal conductivity and numerous electroactive sites (90,91). However, due to van der Waals forces and its inherent laminar structure, rGO tends to aggregate, leading to a decrease in surface area and thus reducing its sensing ability (92,93). The commonly used reducing agents for rGO, such as hydrazine and NaBH₄, are highly toxic and hazardous (94). Chaturvedi et al (95) addressed this issue by reducing GO to rGO and coating it with a biocompatible, nanometer-thick polydopamine (PDA) layer. PDA is rich in functional groups such as amines, imines and catechols, facilitating dense covalent attachment of biomolecules and providing binding sites for metal nanoparticles. Consequently, they engineered a nanocomposite of rGO, PDA and AuNPs and applied it to carbon electrodes to enhance the electroactive surface area and electron transport. Electrochemical analysis using cyclic voltammetry and linear sweep voltammetry revealed a sensitivity of 2.12×10⁻³ mA μM⁻¹ and a response time of 5 sec for target DNA detection at 0.1×10⁻⁷ mM.

PDA thin coatings improve the antifouling properties and cytocompatibility of carbon nanomaterials (96). The adhesive properties of PDA facilitate the attachment of biomolecules to biosensor transducers through physical interactions (97). Polynorepinephrine (PNE), a compound closely related to PDA, possesses additional -OH groups and superior coating uniformity; however, it has rarely been investigated in TB biosensors. (98,99). Bisht et al (81) researched PNE as a coating for rGO and AuNPs in the development of an electrochemical nanobiosensor targeting MTB (Fig. 8). The active rGO, coupled with the reactive quinone groups and AuNPs, synergistically forms a high-performance biosensing platform that facilitates substantial biomolecule loading and delivers an exceptional electrochemical response. The study demonstrated that the PNE-modified system (rGO/PNE/Au) outperforms the PDA-modified counterpart (rGO/PDA/Au) for the development of electrochemical biosensors. The PNE-modified system achieves a markedly higher electrochemical response and offers a surface richer in functional groups, enhancing the loading capacity for biomolecules such as probe DNA. The biosensor demonstrated high sensitivity (2.3×10⁻³ mA μM⁻¹), a low detection limit (0.1×10⁻⁷ μM) and a quick response time of 5 sec.

Paper-based analytical devices (PADs) require minimal training and are highly portable, which is crucial for field testing and point-of-care TB diagnostics (100). Graphene nanomaterials have a large surface area, which provides more active sites for biomolecule adsorption, enhancing the electrochemical properties of sensors (101). They boost the sensitivity and specificity of PADs, allowing for the detection of TB biomarkers at low concentrations. Pornprom et al (78) introduced a PAD biosensor using AuNP-decorated carboxyl graphene (GCOOH) to detect heat shock protein (Hsp16.3), a key TB infection biomarker. The AuNPs enhance the electrochemical properties of the sensor, while the GCOOH, with its numerous binding sites, facilitates direct antibody immobilization through carboxyl groups and primary amines. The PAD sensor specifically recognizes Hsp16.3, requiring only 5 μl sample volume, performed effectively with a detection limit of 0.01 ng/ml and quickly detected TB-infected clinical samples within 20 min.

Unlike other electrochemical sensors, field-effect transistor (FET) biosensors involve semiconductor manufacturing (102). This enables the large-scale production of these sensors, making them ideal for widespread use in assessing infection status, which is the purpose of point-of-care testing (103). Graphene-based field-effect transistors (GFETs) have a low on/off ratio compared with other semiconductor materials because they lack a bandgap. However, the low noise characteristic of GFETs can compensate for this limitation, enhancing their overall performance (104,105). Seo et al (82) designed a GFET biosensor for MTB MPT64 protein detection to construct an effective point-of-care TB testing platform. To efficiently conjugate antibodies, the graphene channels of the GFET were functionalized by immobilizing 1,5-diaminonaphthalene (1,5-DAN) and glutaraldehyde linker molecules. Atomic force microscopy was used to investigate the surface roughness of graphene after functionalization with MPT64 Ab and 1,5-DAN. As shown in Fig. 9, Raman spectroscopy and X-ray photoelectron spectroscopy validated the successful and uniform immobilization of linker molecules on the graphene surface and the subsequent antibody conjugation. The MPT64 antibody-functionalized GFET achieved a detection limit of 1 fg/ml in real-time and demonstrated greater sensitivity and faster detection compared with ELISA.

Single-walled carbon nanotubes (SWCNTs), another popular carbon nanomaterial, are similar in size to biomolecules and have an average diameter of 1 nm (106). They possess low charge-carrier density and high intrinsic carrier mobility, making them ideal for detecting electrostatic interactions and charge transfer during biological processes (107). Since 1998, SWCNTs have been used to fabricate FETs, demonstrating exceptional performance in biosensing due to their distinctive physical characteristics (108). SWCNTs have advantages over graphene, silicon nitride and silicon nanowires as FET functional nanomaterials. Their tiny diameter helps reduce gate leakage and exhibit high conductivity, biocompatibility, charge mobility and stability (109,110). Researchers have constructed SWCNT-based FET biosensors to detect SARS antigens (111), cancer exosomal miRNA (112) and Alzheimer's disease biomarkers (113). The limit of detection of these biosensors is equivalent to advanced techniques such as nucleic acid amplification tests and ELISA.

Wang et al (79) developed a SWCNT-based FET device that was functionalized with an anti-MTB antigen 85B antibody (Ab85B) to detect the MTB-secreted antigen 85B (Ag85B). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/Sulfo-N-hydroxysuccinimide (NHS) coupling linked Ab85B to commercial SWCNT sidewalls via carboxyl groups (Fig. 10A). The Ab85B-SWCNT FET device successfully detected Ag85B in phosphate-buffered saline with a detection limit of 0.05 fg/ml. Furthermore, it effectively identified Ag85B spiked in artificial sputum. Additionally, bovine serum albumin-blocked Ab85B SWCNT FET devices could detect Ag85B in serum, distinguishing TB-positive clinical samples from negative ones within 10 min using a portable Metrohm potentiostat. These results demonstrate the potential practicality of the biosensor for TB diagnosis (79).

FNDs are carbon nanoparticles with nitrogen vacancy defects (114). The ground-state electron spins at the center of these nitrogen vacancies can be optically polarized, resulting in spin-state mixing induced by a time-varying magnetic field (115). Diagnostics of ultrasensitive sub-half-molar disease markers are possible with microwave-modulated spin resonance (116). Le et al (80) developed a spin-enhanced lateral flow immunoassay for TB diagnostics by conjugating FNDs with ESAT6 antibodies (Fig. 10B). This immunosensor demonstrated 100-fold higher sensitivity than traditional AuNPs-based lateral flow immunoassays. The FNDs used in this study were ~100 nm and contained ~10 ppm nitrogen-vacancy centers. By employing a lateral flow membrane strip with a pre-structured 1-mm narrow channel, the detection limit for ESAT6 antigen was ~0.02 ng/ml. This FND-based magneto-optical sensor identified MTB complexes in clinical samples and distinguished TB from NTM. Moreover, the immunosensor is a simple portable device that can be used in point of care and clinics.

AuNPs and AgNPs are user-friendly due to simple conjugation processes (117). By contrast, QDs need complex surface modifications, while MNPs and carbon nanomaterials require sophisticated handling for optimal performance (118,119).

AuNPs and AgNPs are straightforward to synthesize via chemical reduction (120). QD synthesis is more complex, focusing on size and shape control for optical properties (121). MNP synthesis varies by composition and size and graphene involves scalable but costly methods such as chemical vapor deposition (122).

AuNPs are stable for long-term use, while AgNPs are more susceptible to oxidation (123). QDs are photostable and MNPs remain stable in different conditions. Graphene and SWCNTs are stable but prone to aggregation, needing functionalization for improved dispersion (124).

AuNPs and AgNPs easily integrate into biosensors. QDs, despite toxicity concerns, offer tunable fluorescence in biosensing. MNPs are ideal for magnetic separation in assays (125). Carbon nanomaterials are adaptable for electronic and electrochemical biosensors but may require miniaturization for point-of-care use (126).

In summary, the choice of nanomaterial for TB diagnostics is application-specific, balancing manipulation ease, synthesis complexity, stability and adaptability to achieve sensitive, specific and cost-effective biosensors.

Nanomaterials can degrade over time, leading to reduced sensitivity and reliability of the biosensors. Factors such as environmental conditions, storage methods and interaction with biological fluids can affect their stability. Surface modification techniques, such as coating with stabilizing agents such as polyethylene glycol or thiol groups, can enhance the stability of nanomaterials. Additionally, rigorous quality control measures during manufacturing and storage can help maintain the integrity of the nanomaterials (127).

Some nanomaterials, particularly MNPs and QDs, can exhibit toxicity when introduced into biological systems. This can lead to adverse effects on cells and tissues, limiting their use in in vivo diagnostics. Surface functionalization with biocompatible polymers or targeting ligands can reduce toxicity and improve cell uptake. Furthermore, developing biodegradable nanomaterials can mitigate long-term health risks (128).

Biological and chemical components in patient samples can interfere with the detection process, leading to false positives or negatives. Common interferents include proteins, lipids and other biomolecules Advanced sample preparation techniques, such as pre-concentration and purification, can reduce interference. Additionally, designing nanomaterials with specific recognition elements, such as antibodies or aptamers, can enhance selectivity and reduce cross-reactivity (129).

The synthesis and functionalization of nanomaterials can be costly and technically challenging, particularly for large-scale production. High costs can limit the accessibility of these technologies in resource-limited settings. Developing cost-effective synthesis methods, such as green chemistry approaches and scalable manufacturing processes, can reduce production costs. Additionally, optimizing the use of nanomaterials to achieve the desired performance with minimal material usage can help make these technologies more affordable (24,129).