## 4 Discussion  
This work focused on the development of an on-site readout device for the implementation of colorimetric assays and the corresponding assay development. The assay is based on the observation that DNA-AuNP probes remain dispersed within the presence of specific LAMP amplicons that bind to the functionalized particles, but aggregate without DNA or unspecific DNA after the addition of an aggregation promoter (MgCl\({}_{2}\), HCl). The 2-LED-uSpectrophotometer has the capabilities to monitor the progress of a nanoparticle aggregation in real-time by measuring the absorbance of the solution at two selectable and spectrally different wavelengths. The performance of the assay was assessed in terms of specificity, dynamic range, and LOD. For specificity evaluation, _Legionella_ DNA was used as specific and non-specific templates, respectively. We demonstrated the abilities by an application of noble-metal nanoparticles in which the reactions contain complementary DNA sequences (specific amplicon), hybridized with the DNA probe on the surface of the AuNPs, resulting in stabilization. Therefore, the addition of MgCl\({}_{2}\) salt or HCl cannot induce aggregation of the DNA-AuNP probe. As a result, the color of the reaction remains red. Alternatively, the reaction without complementary DNA sequences (NTC, non-specific amplicon) after adding MgCl\({}_{2}\) salt leads to aggregation of the AuNP probes and the reaction turns blue/purple. This approach is highly sensitive and selective compared with others without previous DNA amplification [39]. The DNA-AuNP probe assay is faster than the culture method (60 min vs. 24 h) and the DNA amplification requires only a thermal block or a water bath. LAMP requires less sophisticated instrumentation, no temperature cycling, as well as lower maximum temperatures (only about 60 \({}^{\circ}\)C), and is more robust towards inhibitors of unpurified samples [43; 44]. LAMP can achieve excellent specificity and other advantages in comparison with other DNA amplification methods that were previously mentioned [41]. The spectrophotometric analyses of the synthesized AuNPs show an absorbance peak at 520  nm. Their size and the functionalization success were also confirmed using dynamic light scattering measurements (Zetasizer nano series, Malvern Instruments Limited, Worcestershire, UK) (Figure S2). The developed DNA-AuNP probe assay has a clearly visible detection limit at a DNA concentration of 0.5 pg uL\({}^{-1}\), depending on the LAMP LOD. In addition, the DNA-AuNP probe used in this study had no cross-reactivity with the _Legionella_ isolates and the other bacteria. The results were also consistent with the LAMP methods. It should be pointed out that the hybridization of the specific LAMP amplicons and the DNA-AuNP probe was temperature-independent. The colorimetric step of the assay after LAMP amplification took an 8-15 min response time to fully  
Figure 9: Colorimetric LAMP assay using non-crosslinking method for DNA detection. (**a**) Aggregation assay with specific and unspecific LAMP products using the _lepB_ specific AuNP probe and amplicons (specific) obtained from the LepB primerset as well as amplicons (unspecific) obtained from the dotA primerset. (**b**) Absorption ratio mean values: salt-induced method and corresponding tube images, obtained after 15 min. (**c**) Absorption ratio mean values: pH induced method and corresponding tube images obtained after 15 min. Horizontal line: threshold value. NTC, negative template control. aggregate and obtain a reliable signal. The aggregation process could be monitored by measuring the absorbance at 1 s intervals (530 nm, 430 nm, 630 nm, 690 nm). The instrument fulfills a set of criteria, starting with simplicity and cost-efficiency. It can measure dual wavelengths, including a versatile choice of different wavelengths, and also allows the use of standard disposable cuvettes, preventing cross-contamination. The platform is robust and compact, including an internal referencing system in small size.