Selective Sensing and Imaging of Penicillium italicum Spores and Hyphae Using Carbohydrate–Lectin Interactions

Abstract

The blue-green mold Penicillium italicum is among the most problematic post-harvest plant infections limiting the integrity of citrus and many other crops during storage and transportation, but there is no sensor for its on-site or field detection. We hereby, for the first time, report the development of novel biomolecular sensor for assessing the presence of P. italicum spores and hyphae using carbohydrate-lectin recognitions. Two approaches were developed: (i) lateral tests using standalone poly(amic) acid (PAA) membranes and glass surfaces and (ii) quantitative tests on 96-well polystyrene plates and paper electrodes. In both cases, the surfaces were functionalized with novel derivatized sugar based ligands while staining was performed with gold nanoparticles. Both approaches provided strong signals for 10 spores/mL of P. italicum isolated from experimentally infected lemons as the lowest-reliable concentration. The 96-well plate-based gave the most sensitive detection with a 4 × 10 spores/mL limit of detection, a linear dynamic range between 2.9 × 10 and 6.02 × 10 spores/mL ( R = 0.9939) and standard deviation of less than 5% for five replicate measurements. The selectivity of the ligands was tested against Trichaptum biforme, Glomerulla cingulata ( Colletotrichum gloeosporioides), and Aspergillus nidulans fungi species. The highest selectivity was obtained using the sugar-based gold-nanoparticles toward both the spores and the hyphae of P. italicum. The advanced specificity was provided by the utilized sugar ligands employed in the synthesis of gold nanoparticles and was independent from size and shapes of the AuNPs. Accuracy of the sensor response showed dramatic dependence on the sample preparation. In the case of 5-10 min centrifugation at 600 rpm, the spores can be isolated free from hyphae and conidiophore, for which spiked recovery was up to 95% (std ±4). In contrast, for gravity-based precipitation of hyphae, the spiked recovery was 88% (std 11).

Greener One-Pot Synthesis of Gold Nanoparticle Glycoconjugates Using Functionalized Sugars

“…It should be stated that the limitation of the NMR studies was that the sugar ligands and AuNP-GCs were soluble in only D 2 O, so it was not possible to see the protons on NH, NH 2 , and COOH groups what could give a clue about the formation and stability of AuNP-GCs. In contrast to this, 1 H and 13 C NMR spectra of the sugar ligand and corresponding AuNP-GCs (Figure S4a−d) revealed both hydroxyl groups on sugar moieties and aromatic protons joined the stability of AuNP-GC. Similarly, 2D NOESY NMR spectra (Figure S4a) revealed that aromatic protons of the phenyl ring and -CH 2 -groups on the sugar moiety (in the case of β-lactose, which was glucose) were interacting, which could be another contributor to AuNP-GC stability.…”

“…In the case of the sugar ligands shown in Figure 7, none of them provided stable AuNPs, thus protecting the SPR peak for only ∼1 h. 13 C NMR spectra revealed the loss of a carbon peak joined to a primary amino group and showed new peak formation. Overall, the sugar ligands possessing a primary amino group, a thiol group, and an aliphatic group-substituted carboxyl group did not provide stable AuNP-GCs.…”

“…Functionalization of metallic nanoparticle surfaces for cell− cell, cell−pathogen, and ligand−receptor interactions 1−3 has been reported using peptides, 4,5 amino acids, 6−8 carbohydrate polymers, 9 lipids, 10 sugars, 11−13 antibodies, 14 phages, 15 lectins, 16 and glutathione and flavonoids. 17,18 These metalized surfaces have been explored in biological applications, including biosensors, 13,16,19 drug delivery, 12,20 bioimaging, 21 and vaccine development. 22,23 The multivalent properties of carbohydrate-based ligands have been used to improve the sensitivity and selectivity of the targeted recognition in AuNP glycoconjugates (AuNP-GCs).…”

“…Furthermore, lectins possess carbohydrate recognition domains (CRD) and key surrounding amino acids producing high specificity for sugar residues. It has been reported that modifications of sugars (e.g., group addition) afford ≤1000-fold improvements in carbohydrate−lectin specificity 28,29 when used in biosensors 13 and drug delivery. 30 The current synthetic approaches for AuNP-GCs cannot provide flexible and highly reproducible glycoconjugates that meet the requirement of the target applications.…”

On the Effect of Modified Carbohydrates on the Size and Shape of Gold and Silver Nanostructures

“…Gold (Au) and silver (Ag) nanomaterials have applications in various fields including biomedicine, energy storage, drug development and delivery, sensor technologies, environmental remediation, and electronic displays [ 1 , 2 , 3 , 4 ]. In particular, wet-chemistry-mediated synthesis of these nanostructures are of great interest owing to their flexible design in controlling the size, shape, and surface chemistry [ 5 ].…”