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Introduction

Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB), which affects 25% of the world's population. In 2022, the World Health Organization reported 10.6 million new TB infections, 133 per 100,000 persons and 1.3 million mortalities (1). TB is the second most deadly infectious disease worldwide, surpassed only by COVID-19, with its mortality rate nearly double that of HIV/AIDS (2). Early and accurate TB diagnosis is crucial for its control and management (3). Early discovery improves therapy, limiting illness progression and serious consequences (4). Accurate identification of TB cases can reduce the spread of MTB, especially in densely populated and resource-limited areas, thereby reducing pressure on the healthcare system (5).

Immunological, radiographic and bacteriological methods are used to diagnose TB (6). The tuberculin skin test and INF-γ release assay are simple immunological assays that detect TB within 72 h. However, the window time of the disease, the immune system and experimental methods can cause false positives and negatives (7). Chest X-rays and computerized tomography scans can detect lung abnormalities and track illness progression, but they are less sensitive and specific and cannot distinguish TB from other lung infectious disorders (8,9). Antacid smear microscopy and sputum culture are the main bacterial tests; however, smear microscopy has just 30% sensitivity and mycobacterial culture, the gold standard, takes 2 weeks to provide positive results, during which MTB will spread in the population (10,11). Automated Nucleic Acid Amplification (PCR) Assay System (GeneXpert) can detect MTB and rifampicin resistance in 2 h (12); however, due to the expensive cost of instruments and reagents and the strict environmental requirements of the assay, GeneXpert is challenging to apply in distant and impoverished locations and its sensitivity is still limited for sputum specimens with low bacterial loads (13). Thus, rapid, cost-effective, precise and sensitive TB diagnostic methods are needed.

Nanomaterials have advanced TB diagnostics in recent years (14). Their high surface area-to-volume ratio provides more reaction sites, greatly enhancing detection sensitivity and enabling accurate TB detection even at low bacterial loads (15). Nanomaterial-based detection technologies frequently yield fast outcomes (16). For example, colorimetric reactions or electrochemical biosensors based on gold (Au) and silver (Ag) nanoparticles (NPs) markedly reduce the time required for conventional detection methods (17,18). Nanomaterials can bind to multiple molecules, allowing simultaneous detection of various TB biomarkers (19). Such multifunctional platforms provide comprehensive diagnostic information, enhancing accuracy and practicality (20). For example, AuNPs allow for the attachment of multiple antibodies or oligonucleotides, which can specifically bind to different TB biomarkers (21). Similarly, quantum dots (QDs) can be conjugated with multiple oligonucleotide probes that are complementary to different segments of the TB genetic material (22). This allows for the simultaneous detection of multiple TB genetic markers. The ability to detect multiple biomarkers simultaneously is particularly beneficial in the early stages of TB infection, where the bacterial load may be low and in differentiating TB from other respiratory diseases that have similar symptoms (23). It also aids in the rapid identification of drug-resistant TB strains, which is vital for initiating appropriate treatment strategies (24,25).

Due to the efficiency and simplicity of nanotechnology, diagnostic devices based on nanomaterials are typically compact and easy to operate, making them suitable for on-site testing and resource-limited settings (26). Fig. 1 presents the nanomaterials and corresponding biosensors for TB diagnosis. The current article reviewed the latest research advances in TB diagnostics using nanomaterials, including their mechanisms and functions and analyzes the structure and performance of various novel nano biosensors. The limitations and challenges of nanomaterials in TB diagnosis are also discussed, along with strategies to overcome them. The present review aims to provide researchers with insights for developing safe, rapid and effective TB diagnostic methods.

Figure 1 Nanomaterial-based biosensing strategies for TB diagnosis. TB, tuberculosis; SPR, surface plasmon resonance.

Metal nanomaterials-based diagnostics for TB

Metal nanoparticles improve TB diagnosis by overcoming some traditional drawbacks. Table I presents recent advances in metal nanomaterials-based diagnostics for TB. Due to their optical, electronic and magnetic characteristics, they can be modified with various ligands and detect TB biomarkers at low concentrations (13,27). Moreover, they can be designed to be portable and user-friendly for convenient use at the medical treatment site. This allows for the adoption of decentralized testing in areas with limited resources, making diagnostic assays potentially more affordable (28).

Table I Recent advances in metal nanomaterials-based diagnostics for tuberculosis.

Table I

Recent advances in metal nanomaterials-based diagnostics for tuberculosis.

First author/s, year	Nanomaterials	Detection assays	Target	LOD	Detection Time/Sample type	(Refs.)
Seele et al, 2023	AuNPs	Lateral flow Immunoassays	CFP-10/ESAT-6	7.69/0.063 ng/ml	15 min/Spiked sample	(29)
Kamra et al, 2023	MB-AuNP	Immuno-PCR assay	MPT-64	1 fg/ml	NA/Clinical sample	(30)
Dahiya et al, 2023	MB-AuNP	Immuno-PCR assay	MPT-64/CFP-10	9.9 ng/ml	NA/Clinical sample	(31)
Tripathi et al, 2023	AuNPs	Colorimetric detection	MTB DNA	0.125 ng/ml	3 min/Amplified sample	(32)
Huang et al, 2023	MXene/C60NPs/Au@Pt	Electrochemical sensor	ESAT-6	2.88 fg/ml	NA/Clinical sample	(33)
Patnaik et al, 2024	AgNPs	AgNP aggregation	MTB DNA	4 bacilli	20-25 min/Amplified sample	(34)
Pei et al, 2022	AuNPs	Dark-field imaging	MTB DNA	10 fM	1 h/Spiked sample	(35)
León-Janampa et al, 2022	MNP@Si@ab	sELISA	MTB antigens	0.15 ng/μl (38 kDa, MoeX, Ag85B) 0.31 ng/μl (MPT64, MTC28) 1.25 ng/μl (CFP10, ESAT6)	4 h/Clinical sample	(36)
Zhang et al, 2022	AuNPs	Piezoelectric sensor	16 S rDNA	30 CFU/ml	3 h/Amplified sample	(37)
Xie et al, 2021	NG@Zr-MOF-on-Ce-MOF@Tb	Electrochemical aptasensor	ESAT-6	12 fg/ml	NA/Clinical sample	(38)
Prabowo et al, 2021	AuNP-ssDNA	SPR sensor	MTB DNA	24.5 fM	NA/NA	(39)
Tai et al, 2021	LSG-NF-AgNP	Electrochemical sensor	MTB DNA	10−15 M	NA/Amplified sample	(40)
Azmi et al, 2021	Fe3O4/Au	Sandwich-type immunosensor	CFP10-ESAT6	1.5 ng/ml	2 h/clinical sample	(41)
Gupta et al, 2021	MNPs	Giant magnetoresistance	ESAT-6	1 pg/ml	NA/Clinical sample	(42)
León-Janampa et al, 2020	MNP@Si@NH2	sELISA	Hsp16.3	0.9 pmol	NA/purified sample	(43)

[i] CFP-10, CFP10, culture filtrate antigen, 10 kDa; ESAT-6, early secreted antigenic target-6; MPT64, MTB 64 protein; LOD, limit of detection; NA, not available; MB-AuNP, magnetic bead-coupled gold nanoparticle; MOF, metal-organic framework; NG, nitrogen-doped graphene; Tb, electroactive toluidine blue; SPR, surface plasmon resonance; LSG-NF, graphene nanofiber laser biosensor; MNPs, magnetic nanoparticles; ab, polyclonal antibodies; sELISA, sandwich enzyme-linked immunosorbent assay.

AuNPs-based TB detection

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.

Figure 2 AuNPs-based colorimetric detection of TB DNA Schematic diagram: Two PCR tubes were made using TB primers and PCR mix. The TB DNA template was placed in one tube and the other tube without the TB DNA template. After PCR, AuNPs and ethanol were added. The tube without the DNA template stayed red, while the tube with TB DNA turned purple. Reproduced from (32), Copyright (2023), with permission from Royal Society of Chemistry. AuNPs, gold nanoparticles; TB, tuberculosis.

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.

Figure 3 Procedure of piezoelectric sensor based on AuNPs-mediated enzyme-assisted signal amplification. (A) SEM images of electrodes promoting the growth of AuNPs in HAuCl4 and NADH solutions containing target DNA and Exo III for (Ba) 0 min, (b) 10 min, (c) 30 min and (d) blank control. Reproduced from (37), Copyright (2022), with permission from Elsevier. AuNPs, gold nanoparticles.

AgNPs-based TB detection

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.

Figure 4 Procedure for acpcPNA-induced AgNP aggregation. (A) AgNPs were initially well dispersed by the negatively charged electrostatic repulsion. Positively charged acpcPNA shielded them from electrostatic repulsion, causing silver particles to aggregate and a color reaction to occur. When complementary DNA was present, the specific PNA-DNA interaction replaced the PNA-AgNPs interaction, forming negatively charged PNA-DNA double strands that depolymerized the nanoparticles. In the case of non-complementary DNA, the nanoparticles did not depolymerize and no color change occurred. Reproduced from (54), Copyright (2017), with permission through Creative Commons public use license from Teengam P et al, American Chemical Society. (B) Schematic of CFP10-ESAT6 detection using the portable electrochemical reader. Sputum sample analysis was performed locally with the modified SPGE (circled in red) and a portable reader. Following GP/PANI modification of SPGE, the CapAb was immobilized on its surface to capture the target antigen and the Ab-loaded Fe3O4/Au particle bound to the target and amplified the detection signal. Reproduced from (41), Copyright (2021), with permission from Springer Nature. acpcPNA, PNA with a positively charged lysine modification at its C-terminus; AuNPs, gold nanoparticles; PNA, peptide nucleic acid; CFP10, culture filtrate antigen, 10 kDa; ESAT-6, early secreted antigenic target-6; SPGE, screen-printed gold electrode; GP/PANI, graphene/polyaniline; CapAb, capture antibody; MTB, Mycobacterium tuberculosis.

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.

Magnetic nanoparticles (MNPs)-based TB detection

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.

QDs-based TB diagnostics

QDs are nanoscale semiconductor particles with size-tunable fluorescence, meaning smaller dots emit blue light while larger ones emit red light (19). As fluorescent probes, QDs can mark MTB nucleic acid and are more photostable and less prone to photobleaching than organic dyes (59). The surface of QDs can be modified with various functional groups or nanomaterials to improve their solubility, stability and biocompatibility (60). Table II presents recent advances in quantum dots (QDs) based diagnostics for TB.

Table II Recent advances in quantum dots based diagnostics for tuberculosis.

Table II

Recent advances in quantum dots based diagnostics for tuberculosis.

First author/s, year	Nanomaterials	Detection assays	Target	LOD	Detection time	(Refs.)
Hu et al, 2023	CdTe:Zn2+ QD-NB	Colorimetric assays	MTB DNA	2 copies/μl	55 min	(61)
He et al, 2022	CdTe QD/CoTCPP	Fluorescence quenching	Methyl nicotinate	0.59 μM	4 min	(62)
Hu et al, 2022	Double CdTe QDs/nanoCoTPyP	Fluorescence quenching	rpoB531/katG315	24/20 pM	95 min	(63)
Kabwe et al, 2022	MA-CdSe/ZnS QDs	Visual paper-based lateral flow	Anti-MA antibodies	NA	NA	(64)
Shi et al, 2024	CdTe QD/carbon dots	Fluorescence quantification strategy	IFN-γ/IP-10	0.3/0.5 ag/ml	8 h	(65)
Kabwe et al, 2022	MA-GQDs	Lateral flow tests	Anti-MA antibodies	NA	NA	(66)
Liang et al, 2021	CdTe QDs/Cu-TCPP	FRET	IS6110	35 pM	50 min	(67)

[i] LOD, limit of detection; MTB, Mycobacterium tuberculosis; QD-NB, quantum dot-based nanobeacon; NA, not available; CoTCPP, cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin; nanoCoTPyP, nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H porphine; Mas, mycolic acids; GQDs, graphene quantum dots; FRET, fluorescence resonance energy transfer; TCPP, Tetrakis(4-carboxyphenyl)porphyrin; IP-10, IFN-γ-induced protein 10.

Bakhori et al (68) reported an electrochemical platform based on CdSe/ZnS QDs and silica nanoparticles (SiNPs) to detect TB-specific biomarkers (CFP10–ESAT6). They demonstrated that the active surface area of the CdSe/ZnS QD/SiNPs modified electrode was 4.14-fold higher than a bare electrode. Results indicated a linear calibration curve in the 40-100 ng/ml target concentration range, with a detection limit of 1.2×10−9 g/ml for CdSe/ZnS QD/SiNPs modified electrode and 1.5×10−10 g/ml for SiNPs modified electrode. These results indicated that the CdSe/ZnS QD-modified electrode has superior electrochemical behavior, which improves electron transfer between the electrode and the target.

In fluorescence resonance energy transfer (FRET)-based systems, QDs can provide energy and bind acceptors. When these probes attach to target nucleic acids such as MTB RNA or DNA, structural alterations influence energy transfer efficiency, resulting in quenching or fluorescence changes, allowing quantitative analysis (69,70). This QD quenching technology-based biosensor serves as a fast, sensitive and easy-to-use diagnostic tool (71). Liang et al (67) used carboxyl-modified CdTe QDs to label single-stranded DNA (QDs-DNA) as a fluorescence donor. In their approach, Cu-TCPP (a two-dimensional metal-organic framework) nanosheets were used as the fluorescence acceptor for QDs-DNA. QDs-DNA attached to Cu-TCPP, resulting in fluorescence quenching in the absence of targets. However, when the target nucleic acids were present, QDs-DNA formed with them a dsDNA complex, preserving strong fluorescence (Fig. 5A). The sensor exhibited a linear response from 0.05 to 1.0 nM and a 35 pM detection limit. This QD-based fluorescent technology for clinical sputum analysis achieved high sensitivity and specificity.

Figure 5 QD-based FRET system and colorimetric platform for TB diagnosis. (A) Schematic illustration of FRET-based MTB detection using QDs-DNA(fluorescence donor) and Cu-TCPP (fluorescence acceptor). Reproduced from (67), Copyright (2021), with permission from Elsevier. (B) Graphical representation of the QD-NB-based colorimetric platform for TB diagnosis. Reproduced from (61), Copyright (2023), with permission from American Chemical Society. FRET, fluorescence resonance energy transfer; MTB, Mycobacterium tuberculosis; QD, quantum dot; NB, nanobeacon; TB, tuberculosis.

Hu et al (61) proposed a QD-nanobeacon (NB)-based colorimetric platform for TB diagnosis, where the QD-NB acted as a cleavable substrate and a signal indicator. As shown in Fig. 5B, they conducted recombinase polymerase amplification in the presence of the target DNA and chemically denatured the amplicon for DNA, followed by a multicomponent nuclease (MNAzyme) reaction. The MNAzyme identified the target DNA and hybridized with the QD-NB. Upon adding Mg2+, the QD-NB was cleaved into two DNA fragments, triggering the release of green fluorescence due to the FRET effect of QDs. This QD-NB-based MNAzyme colorimetric assay achieved a detection limit of 2 copies/μl, cost ~$4 in reagents and took only 55 min to complete.

The primary inner filter effect (IFE) is the absorption of excitation light by various chromophores in solution or matrix, while the secondary inner filter effect refers to the absorption of emission radiation (72). He et al (62) found that cobalt-metalized tetrakis (4-carboxyphenyl) porphyrin (CoTCPP) could modulate the fluorescence emission and quenching of QDs through the inner filter effect. Thus, they developed a fluorescent probe based on CdTe QDs and CoTCPP nanosheets to analyze methyl nicotinate in vapor samples of MTB (Fig. 6). CoTCPP and QDs cannot become close enough to access FRET due to electrostatic repulsion. By contrast, the IFE affects QD fluorescence quenching. They used red-emitting QDs as fluorescent signal switches whose fluorescent are quenched by CoTCPP but restored by methyl nicotine. The platform effectively detects methyl nicotinate with a relative standard deviation <3.33%, the detection time was only 4 min and it was linear in the range of 1-100 μM with a detection limit of 0.59 μM.

Figure 6 QDs as fluorescent signal switches to detect MTB. Schematic for (A) vapor sample collection and (B) MTB methyl nicotinate detection based on CoTCPP nanosheets CdTe QDs and CoTCPP. Reproduced from (62), Copyright (2022), with permission from Springer. MTB, Mycobacterium tuberculosis; QD, quantum dot.

With single excitation and multiple emission, QDs could identify numerous MTB markers simultaneously. This multiplexing capacity simplifies the instrumentation and experimental setup and comprehensively explains the infection's presence and severity (73,74). Zhou et al (20) developed an immunosensor to measure latent tuberculosis infection biomarkers (IFN-γ, TNF-α and IL-2) by embedding carbon and CdS QDs on AuNPs and magnetic beads. Then three antibody1-labeled markers were immobilized at three electrode positions to capture the corresponding antigens and simultaneously detected with antibody2 and QD functionalized nanoprobes. Hu et al (63) developed a novel fluorescence biosensor that uses nanocobalt 5,10,15,20-tetra (4-pyridyl)-21H,23H porphine (nanoCoTPyP) and dual QDs to simultaneously detect two drug-resistant genes of MTB, specifically rpoB531 and katG315 (Fig. 7). The green and red QDs were linked to the single strand (ss)DNA probes ssDNA1 and ssDNA2 and combined to form QD-ssDNA probes. These probes interact with nanoCoTPyP through electrostatic forces, π-π stacking and hydrogen bonding, resulting in fluorescence quenching through FRET and photoinduced electron transfer. This biosensor enables the concurrent quantification of the two genes in one test using the distinct emission wavelengths of the dual QDs. Notably, this approach allows for the simultaneous identification of two mutations in the PCR products of multi-drug resistant tuberculosis within a 95-min timeframe.

Figure 7 Schematic Illustration for preparing nanoCoTPyPs with different morphology and the simultaneous detection of rpoB531 and katG315 based on double QDs-ssDNA and nanoCoTPyP. The spherical nano-CoTPyP performed the best quenching and sensing properties. Reproduced from (63), Copyright (2022), with permission from American Chemical Society. nanoCoTPyP, nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H porphine; QD, quantum dot; ssDNA, single-stranded probe DNA.

Carbon-based nanomaterials for TB diagnosis

Carbon-based nanomaterials, such as fullerene, carbon nanotubes, nanodiamonds and graphene, show great potential for TB diagnosis (75,76). These materials can be engineered to detect specific TB biomarkers, even at very low concentrations (77). Recent advances in the use of carbon-based nanomaterials for TB diagnostics are summarized in Table III. Additionally, carbon nanomaterial-based point-of-care testing devices can be portable and easily used, which is beneficial in low-resource TB-endemic areas. making them particularly advantageous in low-resource, TB-endemic regions.

Table III Recent advances in carbon-based nanomaterials diagnostics for tuberculosis.

Table III

Recent advances in carbon-based nanomaterials diagnostics for tuberculosis.

First author/s, year	Nanomaterials	Detection assays	Target	LOD	Detection Time	(Refs.)
Pornprom et al, 2024	AuNPs/GCOOH	Paper-based electrochemical biosensor	Hsp16.3	0.01 ng/ml	20 min	(78)
Wang et al, 2024	SWCNT	FET	MTB-Ag85B	0.05 fg/ml	10 min	(79)
Le et al, 2024	FNDs	SELFIA	ESAT6	0.02 ng/ml	NA	(80)
Bisht et al, 2023	RGO/PNE/Au	Electrochemical sensor	MTB DNA	10−8 μM	5 sec	(81)
Seo et al, 2023	Graphene	GFET	MPT64	1 fg/ml	NA	(82)
Mogha et al, 2018	rGO-PDA-Au NP	Electrochemical genosensor	MTB DNA	10−15 M	5 sec	(83)
Li et al, 2022	AQCA/CMK-3-Ce-MOFs	Electrochemical aptasensor	MPT64	67.6 fg/ml	NA	(84)
Rizi et al, 2021	HAPNPTs//MWCNTs	Electrochemical DNA biosensor	Genome of MTB H37Rv	0.141 nM	NA	(85)
Javed et al, 2021	GO-CHI	Electrochemical genosensor	IS6110	3.4 pM	NA	(86)
Omar et al, 2021	Ni-rGO-PANI	CV-based immunosensor	ESAT-6	1.0 ng/ml	15 min	(87)
Jaroenram et al, 2020	Graphene	Electrochemical genosensor	IS6110	1 pg DNA	65 min	(88)
Kahng et al, 2020	SWCNT	Immuno-resistive sensor	MTB/MPT64	10 CFU/ml/100 ng/ml	30 min	(89)

[i] LOD, limit of detection; GCOOH, carboxyl graphene; Hsp, heat shock protein; SWCNTs, single-walled carbon nanotubes; FET, field-effect transistor; MTB, Mycobacterium tuberculosis; Ag85B, antigen 85B, FNDs, fluorescent nanodiamonds; SELFIA, spin-enhanced lateral flow immunoassay; ESAT-6, early secreted antigenic target-6; NA, not available; PNE, polynorepinephrine; rGO, reduced graphene oxide; GFETs, graphene-based field-effect transistors; MPT64, MTB 64 protein; PDA, polydopamine; NPs, nanoparticles; MPT64, MTB 64 protein; AQCA, anthraquinone-2-carboxylic acid; CMK-3,carbon framework; MWCNTs, multi-wall carbon nanotubes; GO-CHI, graphene oxide-chitosan.

Graphene-based TB detection

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.

Figure 8 Schematic diagram of rGO/PNE/Au nanocomposites synthesis and detection procedure for MTB DNA. Reproduced from (81), Copyright (2022), with permission from Elsevier. rGO/PNE MTB, Mycobacterium tuberculosis; rGO, reduced graphene oxide; PNE, polynorepinephrine.

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.

Figure 9 Graphene functionalization for the TB biosensor. (A) Illustration of surface modification of graphene-based biosensor and the coupling process of MPT64 with 1,5-DAN and glutaraldehyde. AFM photos of graphene (B) before surface modification, (C) following 1,5-DAN treatment and (D) following MTP64 Ab conjugation. (E) Raman spectroscopy and (F) X-ray photoelectron spectroscopy characterized the surface of graphene. Reproduced with permission through Creative Commons Attribution License (CC BY) from Seo G et al (82), Frontiers in Bioengineering and Biotechnology; published by Frontiers, 2023. TB, tuberculosis; MPT64, MTB 64 protein; 1,5-DAN, 1,5-diaminonaphthalene.

Single-walled carbon nanotubes-based TB detection

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).

Figure 10 SWCNT-based FET device and FND-based immunosensor for MTB detection. (A) Schematic representation of SWCNTs modified by EDC/NHS and connected to Ab85B. Reproduced with permission through Creative Commons Attribution 4.0 International License from Wang J et al (79), ACS Sensors; published by American Chemical Society, 2024. (B) Schematic representation of ESAT6 (MTB critical virulence factor) detection by competitive spin-enhanced lateral flow immunoassay. Magnetically modulated fluorescence allows background-free detection. ESAT6 in the sample and test strip compete for the few Ab binding sites on the FND to accomplish competitive detection. Movement of the strip is indicated by a black arrow. Reproduced from (80), Copyright (2024), with permission through Creative Commons Attribution-NonCommercial 3.0 Unported Licence, Royal Society of Chemistry. SWCNTs, single-walled carbon nanotubes; EDC/NHS, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide; ESAT6, early secreted antigenic target-6; FNDs, fluorescent nanodiamonds; TB, tuberculosis.

Fluorescent nanodiamonds (FNDs)-based TB detection

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.

Comparison of nanomaterials for TB diagnostics

It is essential to compare the manipulation, production, stability and adaptation of these materials when selecting them for TB biosensing applications.

Ease of manipulation

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).

Synthesis

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).

Stability

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).

Adaptability

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.

Limitations of nanomaterial-based sensing systems and possible solutions

While nanomaterial-based sensing systems have shown significant advances in the detection of MTB, several limitations and challenges must be addressed to fully realize their potential in TB diagnostics.

Stability and long-term performance

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).

Biocompatibility and toxicity

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).

Interference from biological and chemical components

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).

Cost and scalability

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).

Comparison of different response detection technologies for TB diagnostics

In addition to the properties of nanomaterials, response detection technology plays a crucial role in the analytical performance of biosensors for TB diagnostics. Optical assays, such as those using AuNPs, offer simplicity and cost-effectiveness but may have limited sensitivity and be prone to interference from complex sample matrices (130). Fluorescence assays, often employing QDs, provide high sensitivity and specificity due to their unique optical properties, yet they require specialized equipment and can suffer from photobleaching (131). Electrochemical assays, enhanced by carbon-based nanomaterials such as graphene, are known for their high sensitivity, rapid response and low cost, but are susceptible to electrode fouling and necessitate careful handling (132). Each detection technology presents distinct advantages and challenges and the optimal choice for TB biosensors depends on the balance between sensitivity, specificity, cost and operational simplicity. The development of future biosensors should aim to integrate the strengths of these detection methods to enhance diagnostic reliability and practicality.

Conclusions

In conclusion, nanomaterials for MTB detection may revolutionize TB diagnostics by addressing the inadequacies of clinical approaches. Metal nanoparticles, such as gold and silver, have been employed in colorimetric and electrochemical biosensors to speed up detection. QD-based platforms, such as the QD-NB-based MNAzyme colorimetric assay and the double QDs-ssDNA probe, can detect different TB markers simultaneously and are ultra-sensitive. Carbon-based nanomaterials, such as the graphene-based PAD, can quickly detect MTB in trace specimens. In serum, SWCNT FETs rapidly distinguish TB-positive from negative samples.

Despite the promising research progress reviewed here, limitations and problems remain. For instance, biosensor stability, biocompatibility and long-term performance need improvement. New diagnostic procedures also need substantial clinical validation to assure safety, efficacy and regulatory compliance. In practice, biological and chemical components can interfere with sensors; thus, their anti-interference capabilities must be strengthened. Researchers should improve sensor design, nanomaterial fabrication and data interpretation to overcome such challenges.

Availability of data and materials

Not applicable.

Authors' contributions

LC conducted the overall planning of the review, carried out the literature search and selection process. JZ drafted the core content. HW analyzed and discussed the literature in depth, offering valuable insights and assisting in refining the text. LC performed supplementary literature searches and validations, enhancing the comprehensiveness and accuracy of the review. Furthermore, JZ and LC jointly verified the authenticity of the relevant data points sourced from the reviewed literature. Data authentication is not applicable. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations:

Acknowledgements

Not applicable.

Funding

No funding was received.

References