Randomly positioned gold nanoparticles as fluorescence enhancers in apta-immunosensor for malaria test

Graphical abstract A plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays. Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-04746-9.


S2. Synthesis of gold nanoparticle.
Gold nanoparticles (AuNPs) were synthesized by chemical reduction of gold(III) chloride trihydrate with sodium citrate (Fig. S1) [2]. Particularly, 3.4 mg of HAuCl4·3H20 was dissolved into 50 mL of ultrapure water and the resulting solution was warmed at 150 °C under vigorous stirring. A volume of 6 mL sodium citrate (34 mM) was added during the boiling to lead particle nucleation. A volume of 4.2 mL solvated HAuCl4·3H20 (17 mM) was spiked into the solution after 2 min in order to induce the particle growth. Within few minutes, the colour of the solution went from yellowish to black and then to bright red. The colloidal AuNP solution was cooled down at room temperature for 2 h under vigorous stirring and dark condition.
The procedure included five steps that are schematically shown in

S4. Processing of scanning electron micrographs.
The substrate was morphologically characterized by Zeiss LEO 1550VP field emission scanning electron microscope (FESEM) with a nominal resolution of 1 nm at 20 kV acceleration voltage. The recorded scanning electron micrographs were processed by ImageJ software to retrieve information about the shape and size of nanoparticles and the center-to-center distance. Firstly, raw images ( Fig. S3a shows an example of micrograph acquired at high magnification) were thresholded thereby isolating nanoparticles from the background (Fig.   S3b). Then, adjacent objects were separated by "Watershed" tool implemented in ImageJ (Fig. S3c).
Eventually, perimeter ‫,‬ area ܵ, centroid coordinates and shape descriptors (circularity ‫ܥ‬ = 4πܵ ‫‬ ଶ ⁄ and aspect ratio ‫)ܴܣ‬ were measured by decomposing each object in outline and inner region through "Analyze Particles" tool implemented in ImageJ (Fig. S3d).

S5. Numerical simulation.
The optical response of the 2D AuNP array was simulated by using the finite-difference time-domain (FDTD) method implemented in "FDTD solutions" tool of Lumerical software that solves Maxwell's equations within a Mie problem-like workspace. Such a workspace, consisting of light source, nanostructure, photodetector, and boundary conditions (BCs), was discretized over a mesh (1 nm spatial resolution) so that Maxwell's equation could be numerically solved in the time domain by evaluating the evolution of the electromagnetic field in each sampled volume. An illustration of the simulation workspace is shown in Fig. S4. Linearly xpolarized plane wave traveling along z direction was set to investigate the system, whereas a photodetector was positioned on the opposite side of the workspace to measure the extinction spectrum of the substrate. The

S6. Substrate biofunctionalization through photochemical immobilization technique.
The UV irradiation of Abs produces the selective photoreduction of the disulfide bridge (S-S) in cysteinecysteine/tryptophan (Cys-Cys/Trp) triads giving rise the breakage of Cys-Cys bonds and the formation of free thiol groups (-SH) (Fig. S5a). Such thiol-terminated ends are localized in both Ab Fab fragments and have high chemical reactivity with noble metals. Functionalization by PIT warrants both the covalent tether of Abs onto the AuNP surface and the control over their orientation so to achieve the exposure of one of the two antigen binding sites to the surrounding environment (Fig. S5b) [7].
The UV source consisted of two U-shaped low-pressure mercury lamps (power of single lamp 6 W at 254 nm) that wrapped the quartz cuvette containing the Ab aqueous solution delivering approximately 0.3 W/cm 2 (Fig.   S5c). Such an irradiation intensity was strong enough to warrant the selective thiol group production in both Ab Fab fragments while prevented significant direct photolysis of S-S bonds that poorly absorb at 254 nm [8].

S7. Microfluidic system.
The functionalization was carried out by using a microfluidic circuit consisting of a cell containing the substrate, a 2 mL syringe, and Tygon tubes with a diameter of 1 mm (for both the input and output channel) designed for biological samples (Fig. S6). The volume of the solution in contact with the substrate was approximately 30 μL, whereas the total volume flowing into the circuit was ~200 μL. The syringe was used to repeatedly draw 250 μL of the fresh aqueous solution containing the irradiated Abs (4 draws separated by a time interval of 3 min). Then, the substrate was copiously rinsed by ultrapure water to remove unbound Abs.
Aqueous solution containing BSA proteins (50 μg/mL) was used to block free AuNP surface from any nonspecific adsorption (4 draws of 250 μL separated by a time interval of 1 min).

S9. Processing of fluorescence images.
Image processing and analysis were performed by ImageJ software. Raw fluorescence images exhibited smooth continuous background that was removed by the so-called "rolling ball" algorithm. Such an algorithm locally measured the background by averaging it over a ball around each pixel. The resulting mean values were subtracted from the raw image so that the background was flattened (see Fig. S7). A sphere of 20 pixels diameter represented the best compromise to properly include the largest objects that were not part of the background and to warrant a flat and homogeneous background. Aiming at measuring the whole fluorescence intensity, the images were thresholded at a value slightly higher than the flattened background -so that the resulting background value was zero -and the signals arising from the fluorescence spots were summed.

S10. Protocol for detecting PfLDH in blood.
Once the device is ready to be used, the following steps allow one to measure the PfLDH concentration in human blood: 1) Dilute 10 μL of patient's blood in 990 μL of 25 mM Tris buffer (see Table S1 for the preparation of Tris buffer).
2) Incubate anti-PLDH-functionalized substrate with 1 mL blood sample by gently shaking the bowl for 50 min.
3) Rinse by sequentially dipping the substrate in 25 mM Tris buffer and ultrapure water (three times).
4) Incubate the substrate with 10 mM PBS solution containing 0.1 μM 5-FAM-labelled malaria aptamers by gently shaking the bowl for 30 min in dark condition (see Table S2 for the preparation of aptamerspiked PBS).

5)
Rinse by sequentially dipping the substrate in 10 mM PBS and ultrapure water (three times).
6) Blow-dry the substrate with nitrogen.

7)
Acquire fluorescence images of the substrate at 520 nm (excitation wavelength 490 nm) (see section S8 for more details).
8) Process the raw image and analyse (see section S9 for more details).

S12. Morphological characterization of the substrate.
Nanoparticle diameter was estimated as ‫ܦ‬ = 2ඥܵ π ⁄ while interparticle distance by computing the distance among each centroid and its nearest neighbours. Size distribution is peaked at approximately 31 nm with a standard deviation of 12 nm (Fig. S9a) pointing out no morphological changes were induced during the substrate fabrication process. Right tail of the histogram is due to AuNP clusters as a byproduct of silaneinduced aggregation. Fig. S9b shows the histogram of the centre-to-centre distances whose distribution turns out to be quite broad with 67 nm mean value and 19 nm standard deviation as a result of the randomness of the nanoparticle immobilization onto the substrate. Fig. S9c and S9d shows the distributions concerning the aspect ratio ‫ܴܣ‬ and circularity ‫,ܥ‬ respectively, of the immobilized AuNPs. The mean values ‫ܴܣ‬ തതതത = 1.4 ± 0.4 and ‫ܥ‬ ̅ = 0.90 ± 0.14 convey high regularity in nanoparticle roundness held at macroscopic level.

S13. Functionalization study.
The coverage of AuNPs with a dielectric protein layer leads to a red-shift of the LSPR wavelength up to 4 nm at 50 μg/mL concentration of anti-PLDH. For larger concentrations no change in plasmon resonance was observed due to the saturation of the free gold surface (Fig. S10a). By considering the close-packing arrangement offered by PIT, the number of Abs that can be anchored onto a 30 nm diameter nanosphere is approximately 20 [9]. The excellent covering of the AuNP surface by Abs is also evident by the lack of any significant optical change in the substrate extinction spectrum after the blocking step (blue continuous line in   Only three measurements out of twenty fall outside the 67% confidence interval confirming a good repeatability of the functionalization process. S15. Study of Ab-analyte and Apt*-analyte binding processes. S18. Fluorescence spot analysis.