Facile synthesis of antibiotic-functionalized gold nanoparticles for colorimetric bacterial detection ^†

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Fig. 6

UV-visible spectra illustrating positive detection of bacteria for peni@AuNP (A), cepha@AuNP (B), baci@AuNP (C) and poly@AuNP (D). S. aureus is used as an example in A, B and C, while E. coli is used in D.

Fig. 5 displays several colour variations following introduction of bacteria to the AuNP colloid. In summary, peni@AuNP exhibits a substantial colour change from pink to blue-violet following exposure to S. aureus , while no detectable change was observed upon exposure to P. aeruginosa or E. coli . A similar colour change was observed for cepha@AuNP in the presence of S. aureus and E. coli . This observation is coincident with the known antibacterial activity of penicillin G (benzylpenicillin) and cephalexin, respectively, through attachment to the penicillin-binding proteins in Gram-positive bacteria. ⁴⁶

The baci@AuNP responded to S. aureus , evidenced by a tangible colour change from pink to violet. This result is most likely ascribed to the undecaprenyl pyrophosphate binding capability of bacitracin (a polypeptide antibiotic) that hinders cell wall biosynthesis, ⁴⁷ a feature of some Gram-positive bacteria. On the other hand, SPB variations of poly@AuNP were detected in the presence of P. aeruginosa and E . coli. This transformation can be credited to the selective binding of polymyxin to lipopolysaccharides predominantly present in Gram-negative bacteria. ⁴⁸ All three bacteria implemented in this work produce differing colorimetric patterns, enabling rapid bacterial detection in the presence of an appropriate ATB@AuNP control (in the absence of bacteria).

UV-visible spectroscopy was also used to confirm bacterial detection. As is shown in Fig. 6 , all four of the colorimetric changes perceived by the naked eye were also observed in the corresponding spectroscopic data. A red shift (shift to longer wavelengths) of the λ _max of the AuNP SPB was detected concomitant with the positive visual responses. This wavelength variation can be attributed to AuNP aggregation. This experimental result further substantiates reduced interparticle distance immediately following binding of the ATB@AuNP and bacteria. Assays employing the four ATB@AuNP using lower bacteria concentrations (10 ² CFU mL ⁻¹ ; Fig. S12 ^† ) also afforded a decrease in absorption intensity and red-shifting of the SPB, showcasing the sensitivity and advantage of the described methodology.

To date, few studies have exploited the bacterial detection capability of pharmaceutical-conjugated nanoparticles. The current design focuses on hybrid stability and implementing ATB functional groups that binds to specific sites on the bacterial envelopes. A similar technique has been explored for magnetic nanoparticles, combining nanospecies with fluorescent molecules and vancomycin for the rapid detection low bacteria concentrations. ⁴⁹ However, several drawbacks of this method include low sensitivity of the fluorescence spectroscopic analysis and the multiple step synthetic design. A complimentary report ascribes successful bacteria identification, in part, to a visible AuNP colour change (from red to blue). ⁵⁰ This colour change occurs upon binding of the H2 receptors of the cysteine@AuNP and the bacterial wall. While a step in the right direction, this technique lacks selectivity given the known aptitude of cysteine to bind to a variety interfering molecules present in reaction media. This shortcoming can be addressed through the current methodology given the specificity of the ATB for specific bacteria. Combined with the well-established stability of the ATB under a number of conditions, ⁵¹ the current method offers several advantages compared to previously published routes. The purpose of this work is to ensure that the ATB protectant is able to effectively interact with bacteria upon exposure. While minor modifications of the ATB surface protectants can be expected, as evidenced by FTIR, the primary binding modes of biological molecules (van der Waals and coulombic) ²⁰ are not favourable to significant structural variations. Furthermore, the positive bacterial detection suggests no substantial changes to the active portion of the ATB have occurred throughout the synthetic procedure. However, limitations for the proposed method have yet to be fully recognized. Future directions should be focused on the detection of other strains of clinical importance, such as Mycobacterium – a bacterium that experiences slow growth in vitro . Also, the possibility of false positives due to nanoparticle aggregation in a myriad biological fluids or food liquids should be take into account.