**3.2 Fluorescent probe-based analysis**

 To increase specificity, fluorescent oligo probes such as TaqMan probes and molecular beacons- have been used to monitor and quantify nucleic acid amplification products [68–70]. A fluorescent probe consists of a fluorophore and a quencher covalently attached at 5′ and 3′ ends of a- DNA probe sequence. There are two main types of fluorescent oligo probes, TaqMan probes- and molecular beacons. In TaqMan probes, the fluorescent light of the fluorophore (e.g., FAM) is- absorbed by the quencher (e.g., TAMRA) before amplification; therefore, no fluorescence can be- detected. During amplification, the fluorescent probe hybridizes complementary to the target- sequence, and the DNA polymerase degrades the probe via its 5′-3′ exonuclease activity. As a- result, the fluorescent reporter and the quencher are separated, and the fluorescent reporter is- then detected [71]. This technology has been widely used in real time PCR for medical diagnosis.-

TaqMan probes depend on probe hybridization, polymerase extension, and cleavage of the probes. Molecular beacons, on the other hand, do not require polymerase extension and cleavage activity. Molecular beacons comprise modified stem ends with fluorescent and quencher molecules, a hairpin loop probe sequence (~20–25 bases) and complementary stem sequences (~4–6 base pairs). Before hybridization with the target sequence, the fluorophore on one end of the molecular beacon is quenched by the quencher on the other end of the beacon as the two ends are close together. When the probe hybridizes with the target nucleic acid sequence, the molecular beacon sequence becomes linear. As a result, the fluorophore and the quencher are separated, and the fluorescence is then detected. For example, the putative molecular beacon probes with fluorophore (5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)) and quencher (4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL)) changes conformation and fluoresces spontaneously upon perfect complementarity with the target nucleic acid (**Figure 2)** [72]. This approach is best used to detect ssDNA products produced from SDA.-It can also been multiplexed using multicolored molecular beacons for different targets [69].-

Organic dyes such as rhodamine are conventionally used in fluorescent oligo probes. However, organic dyes have low quantum yield and are easily photo bleached. Other fluorophores such as quantum dots (QDs), silver nanoclusters, upconversion nanoparticles, and corresponding quenchers such as gold nanoparticles and carbon nanomaterials have been used to replace organic fluorophores and quenchers [73] for nuclei acid as well as protein detection [74, 75]. These nanomaterials have high quantum yield and photostability. In addition, they can be simultaneously excited using one wavelength during multiplex detection of various targets. They are promising substitutes of organic dyes in the detection of nucleic acid detection using fluorescent oligo probes.-

Tavares etal. reported an on-chip immobilization of QDs as energy donors in FRET and Cy3-labeled dsDNA target as a receiver for transduction of nucleic acid hybridization, which resulted in rapid quantitative determination of nucleic acid at the fmol level within 7min after target introduction [76]. Silver nanoclusters possess much higher photostability and fluorescence than organic fluorophores and QDs, which have been used to detect influenza specific nucleic acids. Upon hybridization, these DNA-silver nanocluster probes fluoresce up to 500-fold when placed near G-rich nucleic acid targets and exhibited high signal to background ratio [77]. This finding was promising; however, it was elusive how fluorescence increased upon G-rich target detection. It is speculated that upon target binding guanines, G-quadruplex structures may be formed to yield reddish nanoclusters, or serve as electron donors since guanines have lowest oxidation potential comparedwith other nucleotides [78], or otherwise reduce oxidized nanoclusters and render them reddish [79, 80].-

#### **3.3 Lateral flow biosensor-**

Lateral flow biosensor is the most commonly used technology for the point of care testing [81–84]. A test strip consists of four parts: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad. This method uses fiber chromatography material as a solid phase to allow capillary flow of sample solution, followed by the reaction between the analyte in the sample and the target recognition molecules fixed on the nitrocellulose membrane [9] (**Figure 2**). Color development can be obtained through enzymatic reaction, or visually detectable materials such as gold nanoparticles.-

For the detection of nucleic acids, traditional lateral flow biosensor has been modified and- termed nucleic acid lateral flow biosensor. In nucleic acid lateral flow biosensors, antibodies or- antigens are replaced with probe DNAs that are fixed on the test zone and control zone to capture specific targets via nucleic acid hybridization. Based on SDA and aptamers, we developed- a nucleic acid lateral flow biosensor to detect as low as 1cfu/ml of pathogens, 1ppm heavy metals, SNPs, and stem cells [15, 24] (**Figure 2**). This nucleic acid biosensor consists of (i) a specific- capture probe, complementary to one part of the target nucleic acid and conjugated on gold- nanoparticles, (ii) a target-hybridizing probe, immobilized on the nitrocellulose membrane test- zone to capture amplified target sequence, and (iii) a specific nucleic acid probe on control zone- that can hybridize with nanoparticle labeled probe. The hybridization on test line occurs at the- presence of target, while in the absence of target sequence, the test zone does not show up. The- appearance of the control zone shows the assay works properly. This method is fast, specific,- sensitive, and cost effective. With different targeting aptamers and corresponding test zones on- the test strip, multiplexing assay can be developed to detect multiple pathogens [84].-
