**4.4 Hepatitis B**

Hepatitis B virus (HBV) is a viral infection that can be transmitted through contact with infected body fluids (i.e. infected blood) [44, 45]. Hepatitis B surface antigens (HBsAg) and envelope antigens (HBeAg) are recognized clinical indicators used in the diagnosis of HBV [45]. Therefore, the accurate and rapid detection of HBsAg and HBeAg antigens is essential for the clinical diagnosis of HBV. Gold nanoparticles (GNP) coated with streptavidin (SA − GNP conjugate pad), biotin and antibody (Bio − GNP − Ab conjugate bed) have been employed to advance the sensitivity of LFA in the detection of HBV [46]. However, the expensive synthesis of GNPs, the dependence of the LFA on the localized surface plasmon resonance effect of GNPs [47] and their low sensitivity towards HBsAg have resulted in the exploration of QDs for HBV detection [48]. The ease in size tuning, biocompatibility and stable fluorescence has further advantaged QDs over other nanoparticles in LFA development. Several studies have reported the effect of GNPs size on the sensitivity and reproducibility of LFA [45, 46, 49, 50]. For optimum performance of LFA and for enhanced LFA sensitivity, the narrow size distribution of GNPs has been proposed. Kim et al., 2016 [45] reported a reduction in signal intensity with increased GNP size. The group attributed the results to reduced conjugation efficiency of GNP at sizes >42.7 ± 0.8 nm. In another study, Chotithammakul et al., 2021 [49] reported the synthesis of single and mixed-size GNPs for LFA detection of Albumin. Their results showed mixed-sized GNP conjugates exhibited enhanced signals with higher stability and narrower resonance peaks than GNPs with 20 nm sizes. The results suggest that the combination of different sizes of GNP can affect their plasmon resonance. Moreover, in the study, the conjugated mixed-size GNPs - antibodies (anti-BSA) accumulated at the test spot, which improved localized surface plasmon transduction resulting in enhanced optical labelling in the LFA.

#### **4.5 Tuberculosis**

Tuberculosis (TB) is recorded as the deadliest infectious communicable disease caused by *Mycobacterium tuberculosis* and has become a significant public health problem worldwide. Experienced physicians use chest radiographs in clinical practice to detect TB [51, 52]. The clinical diagnosis at reduced time and low cost via LFA is still very underdeveloped for TB diagnosis. Detection of *mycobacterial free tuberculosis* proteins (fprA) using QDs conjugated to two monoclonal antibodies has been reported. The fluorescent QDs were conjugated to the antibodies via a biotin-streptavidin bridge. In this specific LFA, the antibodies were used as capture probes and immobilized onto the test lines of the nitrocellulose strip. The double-antibody sandwich LFA format allowed recombinant fprA protein detection at ~12.5 pg./μL. Compared to traditional immune-based testes (i.e. dot immunobinding assay and Elisa), the antibody labeled QDs LFIA improved the sensitivity, speed (results within 10 min) and simplicity [53]. In another study, Kabwe et al., 2022 [54] explored a paper-based lateral for detecting anti-mycolic acid antibodies. The group used mycolic acid (MA) coated CdSe QDs as a luminescent probe to detect anti-MA antibodies, biomarkers for tuberculosis. The study's results supported test stripbased immunoassays for TB at low-cost and optimum speed.
