Quantum dots-based “chemical tongue” for the discrimination of short-length Aβ peptides

A “chemical tongue” is proposed based on thiomalic acid-capped quantum dots (QDs) with signal enrichment provided by excitation-emission matrix (EEM) fluorescence spectroscopy for the determination of close structural analogs—short-length amyloid β (Aβ) peptides related to Alzheimer’s disease. Excellent discrimination is obtained by principal component analysis (PCA) for seven derivatives: Aβ1-16, Aβ4-16, Aβ4-9, Aβ5-16, Aβ5-12, Aβ5-9, Aβ12-16. Detection of Aβ4-16, Aβ4-16, and Aβ5-9 in binary and ternary mixtures performed by QDs-based chemical tongue using partial least squares-discriminant analysis (PLS-DA) provided perfect 100% accuracy for the two studied peptides (Aβ4-16 and Aβ4-16), while for the third one (Aβ5-9) it was slightly lower (97.9%). Successful detection of Aβ4-16 at 1 pmol/mL (1.6 ng/mL) suggests that the detection limit of the proposed method for short-length Aβ peptides can span nanomolar concentrations. This result is highly promising for the development of simple and efficient methods for sequence recognition in short-length peptides and better understanding of mechanisms at the QD-analyte interface. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-023-06115-0.


Aβ peptides discrimination
The samples for Aβ peptides discrimination were prepared in UV-Star® 96-well plates (Greiner Bio-One GmbH, Kremsmünster, Austria).For each of the investigated analytes (Aβ1-16, Aβ4-16, Aβ4-9, Aβ5-16, Aβ5-12, Aβ5-9, Aβ12-16), the solution of QDs in 50 mM HEPES buffer at pH 7.4 was first pipetted to each well.Next, 2 µL of aqueous solutions of Aβ peptides were added so that the final concentration of QDs and analyte in each sample type was 25 µg/mL and 100 µM, respectively.The volume of all samples was 200 µL.The control samples of QDs were prepared by adding 2 µL of deionized water instead of Aβ peptide solution.Each sample type was prepared in 8 independent replications.The samples were subjected to excitation-emission matrix fluorescence spectroscopy measurements immediately after preparation.

The acquisition of EEM spectra
Excitation-emission matrices (EEMs) of QDs in the presence of Aβ peptides were recorded with a Synergy™ Neo 2 Hybrid Multi-Mode Microplate Reader fluorescence spectrometer (BioTek Instruments, Inc., Winooski, VT, USA).For this purpose, a hand-written protocol was used that relies on recording consecutive emission spectra at increasing excitation wavelengths.
First, samples prepared in a 96-well plate were mixed for 1 min.Then, the emission spectra of each sample were recorded by changing the excitation wavelengths from 250 nm to 500 nm, ∆λex = 10 nm.The range of the recorded emission spectra depended on the excitation wavelength at which the spectrum was acquired to avoid Rayleigh signals.Thus, for λex ∈ (250 nm, 430 nm), the emission was recorded in the range of 450 nm -700 nm, whereas for λex ∈ (440 nm, 500 nm), fluorescence spectra were acquired at λem ∈ (λex + 20 nm, 700 nm).All emission spectra were registered with a constant ∆λem = 1 nm.All experiments were performed at room temperature.

Data analysis
The He-Ne laser (power 4 mW, wavelength 632.8 nm) as a light source.

Spectrofluorometric titration
Spectrofluorimetric titrations of Aβ peptides with aqueous CdCl2 solution were carried out using a FluoroMax®-3 spectrofluorometer (Horiba Jobin Yvon, Longjumeau Cedex, France) at the excitation wavelength of 275 nm (slit widths for emission were 2 nm, whereas for excitation 3 nm) in quartz cuvettes of path length = 1 cm (Hellma GmbH & Co, Müllheim, Germany).The fluorescence intensity was registered for the emission range of 290-400 nm.Salt solutions containing the appropriate level of Cd(II) ions were added to the respective Aβ peptide solution in 50 mM HEPES at pH 7.4, changing the Cd(II): peptide molar ratio from 0.2 to 5.0.

Aβ peptides-induced aggregation of QDs
no changes in the baseline non-specific scattering      Accuracy=97.9%
Figure S. 3. Quantitative analysis of Aβ4-16 performed by QDs-based chemical tongue: (A) PCA score plot showing discrimination of samples at various concentration levels; (B) Hierarchical Cluster Analysis of Aβ4-16 samples in concentration range 0-100 μM (Euclidean distance for mean-centered data was applied); (C) PCA score plot showing discrimination of Aβ4-16 samples at nano-and pico-molar concentration range; (D) QDsbased chemical tongue response towards Aβ4-16 at nano-and pico-molar concentration level, shown as PC1 score (extracted from (C), mean±SD, n=6).The results of two-tailed t-tests as p-values show significant (green, p< α, α=0.05) and insignificant (red, p≥α, α=0.05) differences noticed for various concentration levels when comparing with pure QDs.The limit of detection was found at 1 nM of Aβ4-16.
Figure S. 4. The detection of the individual Aβ peptide (25 µg/mL QDs, 100 µM Aβ, 50 mM HEPES, pH 7.4) in binary and ternary mixtures performed by QDs-based chemical tongue using Partial Least Squares-Discriminant Analysis: (A) composition of 48 studied samples; (B) exemplary EEMs; (C),(D),(E) predicted probability of the presence of Aβ1-16, Aβ4-16, Aβ5-9 in the following samples, respectively.In the case of Aβ1-16 and Aβ4-16 detection was successful for all samples, whereas in the case of Aβ5-9 one false positive was observed (44 th sample marked with an arrow on (E)).