Fluorescent Soybean‑Derived Nano‑Biomass Dots via Ultrasonic Extraction for Sensitive Fe3+ Detection

Background

Luminescent nanomaterials have revolutionised fields ranging from light‑emitting diodes to bioimaging and metal‑ion sensing [1,2,3,4,5,6]. Conventional quantum dots (QDs) deliver outstanding optical properties but suffer from inherent toxicity [13,14]. Consequently, there is a strong drive to identify greener, sustainable luminescent platforms.

Biomass—organic material generated via photosynthesis—offers a renewable source for nanomaterial synthesis. Edible biomass, rich in sugars and proteins, can be transformed into nanodots with minimal environmental impact, ensuring high biocompatibility for biological and environmental uses [15,16]. To date, most biomass‑derived fluorescent nanodots have required high‑temperature carbonisation, limiting scalability and preserving the native structure of the feedstock [22].

Fluorescent nano‑biomass dots (NBDs) have yet to be reported as a green, efficient probe for Fe³⁺ detection. Fe³⁺ plays a vital role in haemoglobin synthesis but excessive levels lead to oxidative damage and organ failure, underscoring the need for sensitive, environmentally friendly sensors [24].

Here, we demonstrate the first ultrasonic‑extracted soybean‑derived NBDs with bright blue emission, a 16.7 % quantum yield, and excellent biocompatibility. Their fluorescence is selectively quenched by Fe³⁺, providing a detection limit of 2.9 µM.

Methods

Materials

Analytical‑grade reagents were used without further purification. Soybeans were sourced from a local supermarket, washed, and stored at room temperature. Calcium chloride, manganese chloride, cupric chloride, cobaltous chloride, lead nitrate, chromium nitrate, ferric chloride, ferrous chloride, cadmium chloride, mercury dichloride, sodium chloride, and zinc chloride were purchased from Aladdin Ltd. (Shanghai, China) and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Synthesis of NBDs

One hundred soybean seeds were rinsed with an ethanol–water mixture to remove surface impurities. They were then immersed in 50 mL distilled water and subjected to ultrasonic agitation for 2 h. The solution’s colour shifted from transparent to dark yellow, indicating nanoscale fragmentation. Following two 3‑minute centrifugation steps at 7,000 rpm, the supernatant was filtered through a 0.22 µm membrane to remove aggregates. The filtrate was frozen at –5 °C for 6 h, lyophilised at –50 °C for 12 h, and the resulting powder was re‑dispersed in water to yield the final NBDs.

Characterization

X‑ray diffraction (XRD) was performed on an X′ Pert Pro diffractometer using a Cu‑Kα source. Transmission electron microscopy (TEM) and high‑angle annular dark‑field scanning TEM (HAADF‑STEM) were carried out on a JEM‑2010 microscope. Fluorescence and UV‑Vis absorption spectra were recorded on an F‑7000 fluorescence spectrophotometer and a UH4150 spectrophotometer, respectively. Fourier‑transform infrared (FTIR) spectra were collected with a Thermo Scientific Nicolet iS10 FTIR spectrometer. X‑ray photoelectron spectroscopy (XPS) was conducted on a Thermo Fisher Scientific ESCALAB 250Xi equipped with an Al‑Kα source.

Photoluminescence Quantum Yield Measurement

Quantum yield (QY) was measured using an F‑9000 spectrofluorometer coupled with an integrating sphere. An aqueous NBD solution (absorptance <0.1) was excited at 370 nm, and emission was recorded from 430 to 450 nm. Reference spectra of pure water were acquired under identical conditions, and QY was calculated using the integrated fluorescence software.

Cellular Toxicity Test

MTT assays evaluated NBD cytotoxicity. HeLa cells were seeded in 96‑well plates, incubated for 72 h in RPMI‑1640 with 10 % fetal bovine serum, and exposed to varying NBD concentrations. Cell viability was assessed by measuring absorbance at 570 nm.

Detection of Fe³⁺

A 1 mL aliquot of NBD solution (3 g L^-1) was mixed with 1 mL of Fe³⁺ solution at different concentrations. After a 1‑minute incubation at room temperature, fluorescence spectra were recorded under 370 nm excitation.

Results and Discussion

Morphology and Chemical Composition

The ultrasonic extraction process (Scheme 1) yields nearly spherical NBDs with diameters ranging from 1 to 3 nm (average 2.4 nm). TEM images (Fig. 1a,b) confirm their amorphous nature, as no lattice fringes are observed. HAADF‑STEM and elemental mapping (Fig. 1d–g) reveal a composition dominated by carbon, nitrogen, and oxygen. Solid‑state ¹³C NMR (Fig. 1h) shows signals at 164 and 170 ppm, indicative of C=O bonds and sp² carbon, respectively.

Schematic illustration of the preparation process of the NBDs from soybeans

TEM images of the NBDs (a) and (b). c The particle size distribution of the NBDs. HAADF image (d) and corresponding elemental distribution mapping of carbon (e), nitrogen (f), and oxygen (g)

XRD (Fig. 2a) displays a broad peak at ~21.5° and a shoulder at ~41.0°, characteristic of amorphous carbon. FTIR spectra (Fig. 2b) show O–H/N–H stretching (~3380 cm^-1), C–H stretching (~2906 cm^-1), and C=O stretching (~1650 cm^-1). Notably, the ~1750 cm^-1 band, attributed to soybean lipids, disappears after ultrasonic extraction, indicating removal of insoluble lipids. XPS (Fig. 2c–f) confirms a composition of 64.33 % C, 32.34 % O, and 2.72 % N, with functional groups including C=O, C–O/C=N, C–C/C=C, pyridinic N, pyrrolic N, C–OH/C–O–C, and C=O. These surface groups confer hydrophilicity and stability in aqueous media.

a XRD pattern of the NBDs. b FTIR spectra of the soybean and NBDs. c XPS survey spectrum of the NBDs. High‑resolution XPS spectra of C 1s (d), N 1s (e), and O 1s (f)

The formation mechanism involves ultrasonic disruption of soybean particles into nanometric fragments, hydrolysis of proteins into peptides and amino acids, and surface functionalisation of the nano‑dots with abundant –OH, –C=O, and –NH groups, which are the primary contributors to fluorescence.

Optical Properties

The NBDs exhibit excitation‑dependent fluorescence: as the excitation wavelength is varied from 320 to 520 nm, the emission peak red‑shifts, enabling spectral tuning (Fig. 3a). In aqueous solution, the NBDs are transparent under ambient light and fluoresce blue under UV illumination (inset, Fig. 3b). PLE spectra (Additional file 1: Fig. S3) reveal an excitation window of 360–420 nm. UV–Vis absorption (Fig. 3b) shows peaks at 270 nm (π–π* of C–C/C=C) and 330 nm (n–π* of C=O/N). Photoluminescence intensity decreases monotonically with temperature (Fig. 3c), consistent with increased non‑radiative recombination. Photostability tests indicate >90 % retention after 6 h of UV exposure, and the NBDs remain stable across 20–80 °C (Fig. 3d).

a Fluorescence spectra of the NBDs with excitation wavelength changes from 320 to 520 nm. b UV‑Vis absorption spectra of the NBDs. c Fluorescence spectra of the NBDs at different temperature, the inset is the plot of the fluorescence intensity of the NBDs as a function of temperature. d Fluorescence intensity and images of the NBD powders under the illumination of a 365‑nm lamp for different duration and those of the NBD powders at different measuring temperature

Cytotoxicity Evaluation

MTT assays with HeLa cells (Fig. 4) show negligible cytotoxicity: cell viability remains >90 % even at 800 µg mL^-1 of NBDs. In contrast, two chemically synthesized carbon dots exhibit viability of 70 % and 67 % at the same concentration, underscoring the superior biocompatibility of biomass‑derived NBDs.

Viability of HeLa cells after 72 h of incubation with different concentrations of NBDs and CDs

Sensing Properties of the NBDs Toward Fe³⁺

Fluorescence quenching by Fe³⁺ is pronounced (Fig. 5a). A linear relationship between F₀/F and Fe³⁺ concentration (0–30 µM) yields an R² of 0.99 (Fig. 5b). The Stern‑Volmer equation gives K_SV = 0.0072 µM^-1, and the limit of detection (LOD) is 2.9 µM—below the U.S. EPA drinking‑water limit of 5.37 µM [24]. Selectivity tests (Fig. 5c) confirm negligible response to Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Cr³⁺, Fe²⁺, Hg²⁺, Mn²⁺, Na⁺, Pb²⁺, and Zn²⁺, while Fe³⁺ produces the most significant quenching. Visual inspection under UV light (Fig. 5d) demonstrates a clear colour change, enabling naked‑eye detection.

a PL spectra of the NBDs in the presence of different concentrations of Fe³⁺. b Calibration curve of the sensor as a function of Fe³⁺ concentration. c Fluorescence intensities of the NBDs in the presence of different ions. d Photographic images of the NBDs solution with different metal ions under indoor and UV illumination

Quenching Mechanism

UV‑Vis absorption spectra (Additional file 1: Fig. S8) show no shift upon Fe³⁺ addition, indicating structural integrity. Fluorescence lifetime measurements (Fig. 6a) reveal a reduction in decay time, suggesting photo‑induced electron transfer from the NBD LUMO to Fe³⁺ d‑orbitals. The quenching is attributed to strong coordination between Fe³⁺ and surface –OH/–NH/–COOH groups, with Fe³⁺ acting as a Lewis acid that facilitates electron transfer (Fig. 6b). This selective interaction accounts for the pronounced sensitivity towards Fe³⁺ over other metal ions.

a Fluorescence decay traces of the NCDs in the absence and presence of Fe³⁺ under excitation at 370 nm and emission at 445 nm. b Schematic illustration for the possible fluorescence quenching mechanism of the NBDs in the presence of Fe³⁺ ions

Conclusion

We have established a green, heating‑free ultrasonic method to produce soybean‑derived NBDs with bright blue emission (16.7 % QY) and exceptional biocompatibility (100 % viability at 800 µg mL^-1). Their fluorescence is selectively quenched by Fe³⁺, delivering a detection limit of 2.9 µM, well below regulatory thresholds. These findings position NBDs as promising probes for biomedical imaging and environmental monitoring.

Abbreviations

FTIR:

Fourier‑transform infrared

HAADF‑STEM:

High‑angle annular dark‑field scanning transmission electron microscopy

LOD:

Limit of detection

NBDs:

Nano‑biomass dots

NMR:

Nuclear magnetic resonance

PL:

Photoluminescence

QDs:

Quantum dots

QY:

Quantum yield

TEM:

Transmission electron microscopy

UES:

Ultrasonic extraction strategy

USEPA:

United States Environmental Protection Agency

XPS:

X‑ray photoelectron spectroscopy

XRD:

X‑ray diffraction