Studying Epigenetic Modifications of DNA Using UV Resonance Raman Spectroscopy

Study of Heavy Metals and Methyl-Mercury in Fungi in Markets of Madrid

Guess Who’s Coming to Inspect R&D?

GC-ICP-MS for Process and Quality Control in Semiconductor Manufacturing

The 2023 Emerging Leader in Molecular Spectroscopy Award

Aaron Acevedo is an assistant editor for LCGC and Spectroscopy at MJH Life Sciences.

Having suitable methods for detecting the quality of food is important, and there are many ways that scientists test for different contaminants. One recent method that has become commonly used in this regard is infrared attenuated total reflection (IR-ATR), which enables the acquisition of evanescent field absorption with almost sample preparation, all while facilitating nondestructive analysis of analytes in any aggregate state (1).

Food quality control concept | Image Credit: © Alexander Raths - stock.adobe.com

, introduced a portable IR-ATR device meant for food analysis, which can be used to analyze analytes deemed notable by food industries (1). The system centered around understanding fatty acid (FAs) composition in lipids; since normal lipid composition characterizes quality of foodstuffs like fish yet are vulnerable to environmental factors like water quality, capture season, and temperature, tracking fatty acids is key to understanding the true characteristics of lipids and how they affect food.

The system was also tested using representatives for mycotoxins and organic solvents. Mycotoxins are harmful secondary metabolites that are associated with fungal contamination. Their presence may induce harmful effects on the health of both humans and livestock, so detecting them is essential to food safety. As for organic solvents, food industries largely use them for constituent extraction from food matrices, though green extraction methods are less popular due to conventional approaches having superior performance. Both substances are essential to food processing, which explains why the IR-ATR system was tested mainly for them in addition to FAs.

The handheld IR-ATR device was tested against conventional laboratory IR-ATR spectroscopy using an FT-IR spectrometer to show the potential of the former system. Three types of model systems were used, each of which had a different sample within: 

NCH) (DMF) dissolved in water, stearic acid (C

) in methanol. All these analytes represent the highlighted class of compounds, having characteristic bands in the mid-infrared (MIR) spectrum.

The comparison between systems confirmed multiple factors in both, including the detection of mycotoxins, the analysis of FAs, and the quantification of organic solvents. Notably, the compact system yielded similar analytical performance characteristics to the standard systems.

However, there is still work to be done before such a system is ready for mass usage. “Future studies aim at analyzing more complex systems including real fish samples, and extracts from various cereals containing fungal contaminants/mycotoxins along with advanced data analysis approaches for the development of label-free rapid screening methods,” the scientists wrote in the study (1).

(1) Fomina, P.; Femenias, A.; Hlavatsch, M.; Scheuerman, J.; Schäfer, N.; Freitag, S.; Patel, N.; Kohler, A.; Krska, R.; Koeth, J.; Mizaikoff, B. A Portable Infrared Attenuated Total Reflection Spectrometer for Food Analysis. 

https://doi.org/10.1177/00037028231190660

Articles

A team of scientists created a portable infrared attenuated total reflection (IR-ATR) spectrometer and tested its food analysis capabilities against similar systems.

Portable Infrared Attenuated Total Reflection Spectrometer Tested for Food Analysis

In this study, 48 different wild and cultivated mushrooms were purchased in local markets in Madrid and provided by trusted specialists from the Mycological Society of Madrid. Cadmium, mercury, and lead were determined by inductively coupled plasma–mass spectrometry (ICP-MS) in samples previously digested in a microwave oven. Methylmercury (MeHg) was determined by liquid chromatography separation (HPLC) combined with ICP-MS after microwave-assisted extraction. Regarding the cadmium and lead contents, all the samples complied with the maximum permitted limits. For mercury, a single sample exceeded the maximum value established in Regulation (EU) 2018/73 for wild mushrooms. In the case of methylmercury, the highest amount represents 13% of the total mercury in one sample. In the rest of the cases, the concentration found was negligible.

Historically, fungi have been classified as inferior plants in the division 

 because of their similarities to other members of that group (no roots, no leaves). However, later studies demonstrated unique features that led to the formation of the new kingdom, the Kingdom Myceteae, which includes microscopic and macroscopic species of fungi. Mushrooms are described as large multicellular fungi with stems and caps. According to Chang and Milles, mushrooms can be simply defined as “macro fungi with a distinctive fruiting body and large enough to be seen with the naked eye and to be picked by hand” (1).

Edible mushrooms have always been a popular food in diets around the world. Their texture and flavor make them highly appreciated in the kitchen. Consuming mushrooms brings multiple benefits because they are rich in protein, dietary fiber, vitamins and minerals, and low in fat. In addition, they contain antioxidant compounds such as polysaccharides, terpenoids, and polyphenols, all of which have a positive impact on health (2,3)

However, the scientific literature shows that mushrooms can accumulate heavy metals present in the soil. The fungal mycelium plays an essential role in this, which spreads over the available area and incorporates metal ions into its cytosol under suitable conditions. Mushrooms are able to accumulate higher concentrations than plants because, unlike their roots, their mycelium develops mainly horizontally, normally occupying the most superficial part (5–10 cm) (4). The mycelium contains the highest concentration of heavy metals (5). Although this characteristic makes them extremely useful as bioindicators of contaminated areas (6), edible mushrooms may pose a risk to consumers if the heavy metal content exceeds reference levels.

The main factors that influence the bioaccumulation of heavy metals in these organisms are environmental factors, which are the concentrations of metals in the soil, the pH, the amount of organic matter and contamination by atmospheric deposition, and intrinsic fungal factors of the species, such as its structure, biochemical composition, decomposition activity, and development of mycelium and fruit bodies (7,8).

Among the heavy metals, it is worth mentioning mercury, cadmium, and lead, which are considered toxic elements for human health by the European Food Safety Authority (EFSA).

Mercury is a toxic heavy metal that can bioaccumulate not only in marine organisms (mainly swordfish and sharks), but also in fungi. There has always been significant concern about its exposures, intakes, and toxicity. In the environment, mercury can be found in three different chemical forms: elemental/metallic mercury (Hg

), and organic mercury (organometallic compounds where one or two alkyl/aryl substituents are attached to the mercury atom). These forms present different bioavailabilities and toxicity, with methylmercury being the most dangerous in the food chain (9).

To perform an accurate risk assessment, it is necessary to collect as much information as possible on the presence of these forms in food because exposure to methylmercury can cause harm by interacting with the sulfhydryl (-SH) groups distributed throughout the body, causing the well- known Minamata disease (6).

The central nervous system is the most important target of methylmercury, which can cause different symptoms, including sensory disturbances in the hands and feet, ataxia, and visual impairment. In addition, organic mercury can reduce the activity of natural killer (NK) cells and generate free radicals. Acute inorganic mercury poisoning affects the gastrointestinal tract and kidneys, causing severe diarrhea, vomiting, and renal tubular necrosis with anuria (6).

In the 2012 EFSA opinion, the tolerable weekly intake (TLWI) for methylmercury was updated to 1.3 μg/kg bw and 4.0 μg/kg bw for inorganic mercury, which is the maximum amount that a person can ingest weekly throughout their lives without adverse effects (9).

European legislation establishes maximum Hg contents in mushrooms. Commission Regulation (EU) 2018/73 distinguishes between cultivated and wild mushrooms, establishing maximum limits of 0.050 mg/kg and 0.50 mg/kg of Hg, respectively. It also establishes a maximum limit of 0.90 mg/kg in 

Cadmium is also a heavy metal that poses serious risks to human health. Until recently, it has not been possible to demonstrate that cadmium has any physiological function, so its main interest lies in its high toxicity in humans, especially in its inorganic forms (11).

Various studies have shown that exposure to cadmium through diet can cause harmful effects on health, mainly in the kidney, spleen, and liver where it accumulates (12). In addition, it is currently classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1), as an association between its intake and the risk of postmenopausal endometrial cancer has been demonstrated. As a result of its ability to induce oxidative stress, it can also affect male fertility by decreasing sperm quality (13).

Prolonged ingestion of cadmium also poses a serious health risk, by causing Itai-Itai disease, the best-known case of which was the mass poisoning that took place in Japan in the last century. This disease is characterized by creating bone damage due to osteoporosis and osteomalacia, in addition to severe kidney damage (14,15). Derived from a comprehensive toxicological assessment, EFSA has established a tolerable weekly intake (TSI) for cadmium of 2.5 μg/kg body weight (16).

In 2023, the maximum cadmium content in mushrooms has been updated by Regulation (EC) No 2023/915, establishing the maximum limit at 0.050 mg/kg for cultivated mushrooms, at 0.15 mg/kg for the mushrooms 

 (oyster mushroom), and 0.50 mg/kg in wild mushrooms (17).

Finally, lead is another heavy metal to consider. Lead contamination arises primarily from the environment, through atmospheric lead through deposition on agricultural crops, from water, or from food processing, handling, and packaging. Although lead exists in both organic and inorganic forms, only inorganic lead has been detected in food (18). When lead is ingested, it is distributed to certain organs such as the brain, kidneys, liver, and bones, causing tissue damage. After several weeks, most of it moves to the bones and teeth, where it can be stored for decades (19,20).

Accumulated lead can return to the blood, reaching vital organs, resulting in episodes of broken bones in the elderly. During pregnancy, accumulation of lead can lead to possible effects on the fetus and lactation (20).

Lead poisoning produces adverse effects at the central nervous system level, which can affect brain development and cause behavioral changes such as reduced attention span and increased antisocial behavior. In addition, it can lead to anemia, hypertension, renal failure, immunotoxicity, and toxicity to reproductive organs (19).

Due to the lack of sufficient scientific information, in 2010 the existing toxicological safety threshold for lead was withdrawn, and a new one could not be determined. Therefore, there is currently no recommended tolerable intake for lead (21).

As of this year, the maximum lead content in mushrooms has been updated by Regulation (EC) No. 2023/915, establishing the maximum limit at 0.30 mg/kg for 

 (shiitake mushroom) mushrooms, and 0.80 mg/kg in wild mushrooms (17).

The main objective for this study is to provide information on the content of cadmium, lead, mercury, and methylmercury in both cultivated and wild mushrooms and to compare the results obtained with the limits established in current legislation. For this, a total of 48 mushrooms were obtained and analyzed.

For the analysis of cadmium, lead, and total mercury, the inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies model 7900) was used. In addition, an integrated sample introduction system (ISIS 3) and an automatic sampler (SPS 4) were used in the determination together with the ICP-MS. The operating parameters of the ICP-MS analysis are shown in Table I.

Methyl-mercury determinations were performed using a liquid chromato- graph (HPLC) (Agilent Technologies 1200 Infinity), consisting of a model 1260 Quaternary Pump VL, a model 1260 ALS injector, and a model 1260 TCC column compartment. This HPLC system is coupled to the ICP-MS described above. The characteristics are detailed in Table II.

For digestions and extractions, a DigiPREP Jr digestion block from SCP Science with 24 positions was used. The volumetric material used for all the solutions were DigiTubes, polypropylene tubes single-use, except in the case of MeHg analysis, which opted for centrifuge tubes, also single-use, VWR Metal Free, 15 mL, and volumetric flasks of 1 L and 200 mL capacity. To take the cor

responding aliquots, Eppendorf brand micropipettes were used.

For centrifugation of the samples, a Lisa refrigerated centrifuge of AFI groups was used.

For the preparation of the reagents, dilutions, washing, and rinsing solution of the work material, deionized water (H

O) of Class Type I (according to the UNE-EN-ISO 3696 Standard) was used, purified with the Milli-Merck’s Q Comprehensive. Nitric acid (HNO

, 65%) from Scharlau was distilled in the laboratory using Savillex equipment, model DST-1000. Suprapur hydrochloric acid for trace analysis (HCl, 37%) supplied by Scharlau and hydrogen peroxide (H

Regarding the ICP-MS, the tuning solution containing 1 μg/l Ce, Co, Li, Mg, Tl, and Y was used. Finally, argon and helium (99.999%) for plasma generation and interference removal.

Standards of 1000 mg/L of Cd (Scharlau), Pb (VHG-Labs), Hg (Scharlau), and MeHgCl (VHG-Labs) were also used. For the determination of cadmium, lead, and total mercury, the intermediate solutions and the calibration curve were prepared with a 2% HNO

As an internal standard, a 0.4 mg/L iridium and rhodium solution (from a 1000 mg/L Scharlau standard) was prepared in a 2% HNO

, 1% HCl, and 5% acetic acid solution for the determination of cadmium, lead, and total mercury. To stabilize the signals, 0.4 mg/L Au from SCP Science was added. No internal standard was used for the methylmercury speciation method.

The composition of the mobile phase was 1 g/L L-cysteine/20 mM CH

/20 mL MeOH in deionized water, with a pH of 6.9, using L-cysteine from Acros Organics (minimum purity 98%), ammonium acetate (CH

) from PanReac AppliChem (96% purity), and methanol (MeOH) from Scharlau (GC residue analysis grade, 

In the case of the mercury speciation analysis, the solution used for the extraction was 5 g/L L-cysteine/0.5% HCl, and the solution used for the preparation of all the intermediate solutions and the calibration curve was the mobile phase.

The samples were homogenized by crushing with a mincer, until a representative sample of the product to be tested was obtained. Two different methods were carried out in this study. Both represented in Figure 1.

FIGURE 1: Schematic diagrams of the different methods (total heavy metals and methylmercury) used for this study.

Regarding the total analysis of mercury, cadmium, and lead, first 0.5 ± 0.05 g of sample were weighed in a DigiTube, then 3 mL HNO

 were added, and the samples were digested in the DigiPrep afterwards. Once the digestion was complete and the samples had reached room temperature, they were made up to volume in 50 mL with deionized water. The sample collection were analyzed by ICP-MS, samples with high analyte contents that were diluted previously.

For methylmercury speciation, 0.2 ± 0.02 g of sample was weighed into a DigiTube, 10 mL of extraction solution (1 g L-cysteine; 5% HCl) was added, and extraction was performed on the DigiPrep for 30 min at 60 

C (Figure 2). Once the process was finished, the samples were allowed to cool to room temperature, transferred to 15 mL tubes, and centrifuged for 10 min at ≥ 4000 rpm and 4 

C. Subsequently, the supernatant was filtered using 0.45 μm pore size filters and placed in 1-mL polypropylene vials to be injected into the HPLC system.

FIGURE 2: Temperature profile for methylmercury speciation. For sample preparation, 0.2 ± 0.02 g of the sample was weighed into a sample tube, 10 mL of extraction solution (1g /L-cysteine; 5% HCl) was added, and extraction was performed for 30 min at 60 °C.

Figure 3 shows a chromatogram where you can see the separation of the peaks obtained, where the first corresponds to Hg, with a retention time of 2.384 min, and the second to MeHg, with a retention time of 3.868 min.

FIGURE 3: The chromatogram illustrates the separation of the peaks obtained, where the first corresponds to Hg, with a retention time of 2.384 min, and the second to MeHg, with a retention time of 3.868 min.

For the calibration curves used in the quantification of the analytes, the 

-axis represents the value of the ratio, calculated as the number of counts of the analyte divided by the number of counts of the internal standard, whereas the 

-axis represents the concentration in μg/L. In the case of MeHg, the 

-axis represents the number of counts per area of the chromatographic peak.

Figures 4, 5, 6, and 7 show the calibration curves used in the quantification of the samples.

FIGURE 4: Calibration curve (with statistics) used in the quantification of cadmium (Cd).

FIGURE 5: Calibration curve (with statistics) used in the quantification of lead (Pb).

FIGURE 6: Calibration curve with statistics used in the quantification of mercury (Hg).

FIGURE 7: Calibration curve with statistics used in the quantification of methylmercury (MeHg).

The test method used for the determination of heavy metals meets the criteria of Regulation (EC) 333/2007 of the Commission of March 28, 2007, which establishes the sampling and analysis methods for the control of levels trace elements and process contaminants from food products and their modifications. The validation was carried out according to the requirements of the UNE/EN-ISO 17025 Standard for Testing Laboratories, and it is accredited by the National Accreditation Entity (ENAC). For MeHg determination, the test method is in the accreditation process.

A total of 48 wild and cultivated mushrooms were acquired and analyzed. All of them were purchased in local markets in Madrid and provided by trusted specialists in the manual collection of mushrooms from the Mycological Society of Madrid.

The results obtained in the study of cadmium, lead, and total mercury of the 48 samples analyzed are shown in Table III.

Regarding the cadmium and lead contents, all the samples comply with the maximum permitted limits, and do not seem to pose a risk to the population.

Regarding the total mercury content, it should be noted that a single sample (

 with a content of 0.97 mg/kg) exceeded the maximum value established in Regulation (EU) 2018/73, set at 0.50 mg/kg for wild mushrooms. No sample of 

 reached the maximum allowed limit of 0.90 mg/kg for said species. In the case of samples of cultivated mushrooms, it is observed that their Hg content is less than 0.050 mg/kg established by the aforementioned regulation.

According to the data obtained, it can be observed that cultivated mushrooms have significantly lower concentrations of metals than wild mushrooms.

The results of the methylmercury determination are shown in Table IV. In this case, only the samples with a significant total mercury content were analyzed. Speciation was performed in the 10 samples that exceeded 0.075 mg/kg of total Hg, all of them wild. The highest amount of MeHg found was 0.12 mg/kg in a sample of 

, which represents 13% of the total mercury in the sample.

It can be determined that the test method used for the analysis of Cd, Hg, and Pb by acid digestion and determination by inductively coupled plasma–mass spectrometry is considered a valid option to carry out a study, such as the one presented here, obtaining results in accordance with the requirements of ISO 17025.

In the same way, the coupling of a liquid chromatograph to an ICP-MS is a recommended method to perform the chromatographic separation of the different mercury species, and to be able to achieve the methylmercury analysis. This method is in the accreditation process.

After determining the content of cadmium, lead, and total mercury and the speciation of methylmercury in the samples analyzed, it can be concluded that the amounts found in the case of cultivated mushrooms are lower than the limits established in European legislation, whereas in the case of wild mushrooms, a result was obtained above the established limits, which may mean a risk to the population.

The values obtained by performing the mercury speciation reveal that the mercury content in the mushrooms is almost entirely inorganic mercury and the amounts of methyl mercury found are practically negligible, becoming, in the worst case, less than 13% of total Hg.

We would like to thank the Mycological Society of Madrid for its invaluable role in providing us with samples of wild mushrooms in order to develop the study of their metal content.

Mushroom: Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact

(2) Gupta, S.; Summuna, B.; Gupta, M.; Annepu, S. K. 

; Springer International Publishing AG, 2018; pp 1815–1847.

(3) Heleno, S. A.; Barros, L.; Martins, A.; et al. Chemical Composition, Antioxidant Activity, and Bioaccessibility Studies in Phenolic Extracts of Two Hericium Wild Edible Species. 

(5) Berthelsen, B. O.; Steinnes, E. Accumulation Patterns of Heavy Metals in Soil Profiles as Affected by Forest Clear-Cutting. 

(6) Bernhoft, R. A. Mercury Toxicity and Treatment: A Review of the Literature. 

(7) García, M. A.; Alonso, J.; Fernández, M. I.; Melga, M. J. Lead Content in Edible Wild Mushrooms in Northwest Spain as Indicator of Environmental Contamination. 

(8) Lalotra, P.; Gupta, D.; Yangdol, R.; Sharma, Y. P.; Gupta, S. K. Bioaccumulation of Heavy Metals in the Sporocarps of Some Wild Mushrooms. 

https://www.efsa.europa.eu/en/efsajournal/pub/2985

(10) European Commission. Commission Regulation (EU) 2018/73 of 16 January 2018 Amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as Regards Maximum Residue Levels for Mercury Compounds in or on Certain Products. 

https://eur-lex.europa.eu/collection/eu-law/consleg.html

(11) Godt, J.; Scheidig, F.; Grosse-Siestrup, C.; et al. The Toxicity of Cadmium and Resulting Hazards for Human Health. 

(12) Kalač, P.; Svoboda, L.; Havlíčková, B. Contents of Cadmium and Mercury in Edible Mushrooms. 

(13) Ciobanu, C.; Şlencu, B. G.; Cuciureanu, R. Estimation of Dietary Intake of Cadmium and Lead through Food Consumption. 

(14) Satarug, S. Dietary Cadmium Intake and Its Effects on Kidneys. 

(15) Inaba, T.; Kobayashi, E.; Suwazono, Y.; et al. Estimation of Cumulative Cadmium Intake Causing Itai–Itai Disease. 

(16) Cadmium in Food—Scientific Opinion of the Panel on Contaminants in the Food Chain. 

(17) European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No. 1881/2006. 

Evaluation of Certain Food Additives and Contaminants: Seventy-Third Report of the Joint FAO/WHO Expert Committee on Food Additives

; WHO Technical Report Series; 960; Geneva, Switzerland, 2011.

(19) World Health Organization. Lead Poisoning and Health. 2022. 

https://www.who.int/news-room/fact-sheets/detail/lead-poisoning-and-health

(20) Agency for Toxic Substances and Disease. Public Health Statement Lead CAS# 7439-92-1. 

https://wwwn.cdc.gov/TSP/PHS/PHS. aspx?phsid=92&toxid=22, 2007

(21) Scientific Opinion on Lead in Food. 

 is with the Public Health Laboratory, in Madrid, Spain. Direct correspondence to: 

The bioaccumulation of heavy metals can lead to disastrous effects on the environment and human health. Here, ICP-MS was applied to study 48 different mushrooms collected at local markets in Madrid, Spain, showcasing its effectiveness at detecting heavy metals in edible fungi.

This year’s Emerging Leader in Molecular Spectroscopy Award recipient is Dmitry Kurouski, an assistant professor of chemistry at the Texas A&M University in College Station, Texas. From his early research days as a graduate student at State University of New York in Albany, Kurouski’s research has emphasized the development and application of innovative Raman spectroscopy methods for noninvasive, nondestructive analyses of biological materials.

. Direct correspondence about this article to 

Dmitry Kurouski is an assistant professor of chemistry at Texas A&M University who has made significant contributions to the field of molecular spectroscopy. His groundbreaking work has earned him the 2023 Emerging Leader in Molecular Spectroscopy Award.

Kurouski’s research pioneered the development of innovative Raman spectroscopy–based sensing approaches that can be used for noninvasive, nondestructive analysis, including confirmatory diagnostics of biotic and abiotic stresses in plants. His findings demonstrate that Raman spectroscopy can be used for the identification of viral, fungal, and bacterial diseases in many plant species. He has also developed Raman methods for diagnostics of plant deficiencies in micro and macro element composition; his work has also demonstrated the potential for Raman spectroscopy–based phenotyping of plants.

 Magazine, recognizes the achievements and aspirations of a talented young molecular spectroscopist, with recipients being selected by an independent scientific committee. The award will be presented to Kurouski at the SciX 2023 conference held October 8–13 in Sparks, Nevada, where he will be honored in a symposium.

Using Raman Spectroscopy for Complex Applications

Kurouski received his PhD in 2013 from SUNY Albany, State University of New York, working with Professor Igor K. Lednev. While working on his PhD, Kurouski developed a deeper understanding of Raman spectroscopy for complex applications.

His early work has led to cutting-edge optical nanoscopic approaches that are based on tip-enhanced Raman spectroscopy (TERS) and atomic force microscopy (AFM) infrared (IR) imaging. His research group now utilizes this nano-Raman-nano-IR imaging approach to reveal the structural organization of amyloid oligomers and protein aggregates that are linked to many neurodegenerative diseases. He has also discovered that the structure and toxicity of amyloid oligomers can be uniquely altered by lipids. These findings suggest that irreversible changes in lipid profiles of organs may be the 

underlying biophysical cause that triggers formation of toxic protein species, which in turn are responsible for the onset and progression of amyloidogenic-type diseases. He was recently awarded a highly prestigious $1 million Maximizing Investigators’ Research Award (MIRA) from the National Institute of Health (NIH) to investigate the effect of lipids on the structure and toxicity of amyloid oligomers.

Kurouski and his research group have made significant progress in understanding the physics of hot carriers and plasmon-driven catalysis on mono- and bimetallic nanostructures. These findings discovered by his group have allowed for elucidation of the underlying physical cause of unique reactivity and selectivity in plasmon-driven catalysis, specifically on gold-platinum and gold-palladium bimetallic nanostructures.

The end of the year celebration party of the Kurouski group in College Station on May 2023. Left to right: Michael Lynn, 2nd year PhD student; Dr. Abid Ali, Postdoctoral Fellow; Aidan Holman, undergraduate student; Axell Rodriguez, undergraduate student; Tianyi Dou, 4th year PhD student, Isaac Juarez, 2nd year PhD student, Luke Osborne, undergraduate student; Professor Dmitry Kurouski; and Kiryl Zhaliazka, 1st year PhD student.

Kurouski’s group developed and deployed Raman spectroscopy for diagnostics of structural and metabolic changes in plants that can be used for confirmatory detection and identification of abiotic stress. The researchers developed spectroscopic libraries that, together with a hand-held Raman spectrometer, could be used to detect and identify nitrogen, phosphorus, and potassium deficiencies in rice. It was also demonstrated that Raman spectroscopy can be used for pre-symptomatic diagnostics of medium and high salinity stress in plants. Together, with more than 40 research reports on this topic, this work showed the emerging potential of Raman spectroscopy in agriculture. These findings are summarized in a review published by the Kurouski group last year.

Kurouski and his research team also have used nano-Raman-nano-IR imaging approaches to unravel the structural organization of viruses. Kurouski found that TERS was capable of resolving protein secondary structure, charge profile, and amino acid composition of the viral capsid with sub-nanometer spatial resolution, providing unprecedented insights into the organization and properties of viruses. At the same time, AFM-IR allowed for the elucidation of structural organization of the nucleic acids in viruses. This work demonstrated the complementary measurements of AFM-IR and TERS in probing depth and chemical information for challenging applications. With the development of confirmatory approaches for highly accurate identification of viruses, Kurouski’s research has significant implications for improving diagnostics and treatment of viral diseases. His groundbreaking work in the field of molecular spectroscopy has already made a significant impact on the scientific community and has the potential to revolutionize many aspects of modern medicine and agriculture.

Professor Kurouski (first on the left) discusses with Dr. Abid Ali (center) and two undergraduate students (Luke Osborne and Joseph Wilson), the strategies for the expression of alpha-synuclein, a protein that is linked to Parkinson disease.

Applying SERS and TERS for Characterization of Biological Systems

In his early work, Kurouski and coworkers used surface- and tip-enhanced Raman spectroscopy (SERS and TERS) as powerful tools for structural characterization of biological systems at the single-molecule scale (1).
However, enhanced Raman spectra of biological specimens often lack the amide I band, which is commonly used as a marker for the interpretation of secondary protein structures.

, the authors investigate the cause of this phenomenon for native insulin and insulin fibrils using both TERS and SERS, comparing these spectra to the spectra of well-defined homo-peptides. The results show that the appearance of the amide I Raman band is not determined by the protein aggregation state, but rather by the size of the amino acid side chain. Peptides with small side chain groups exhibit an intense amide I band in almost all acquired spectra, whereas those with bulky side chains, such as tyrosine and tryptophan, exhibit 

the amide I band in only a fraction of the acquired spectra.

This work provides new insights into the interpretation of Raman spectra of biological specimens and has important implications for the application of SERS and TERS in the study of protein structure and aggregation. The findings have the potential to improve our understanding of protein folding and aggregation mechanisms, as well as aid in the development of new diagnostic tools for protein misfolding diseases.

Professor Kurouski (standing on the right) discusses results of kinetics of protein aggregation with undergraduate fellow Luke Osborne.

The chemical composition of materials at the nanoscale can be challenging to analyze and understand using traditional analytical techniques. To address this issue, the integration of infrared (IR) and Raman spectroscopy with scanning probe methods has resulted in the development of new nanospectroscopy paradigms, such as photothermal induced resonance (PTIR), also known as AFM-IR, and tip-enhanced Raman spectroscopy (TERS). In an article published in 

, Kurouski and co-authors provide a detailed overview of the fundamentals and recent advances of AFM-IR and TERS, as well as complementary features (2).

The article highlights how the recent innovations in AFM-IR and TERS have expanded its applications in various fields, such as materials science, nanotechnology, biology, medicine, geology, optics, catalysis, and art conservation. Although AFM-IR and TERS were initially developed independently for different applications, the recent advancements have pushed its performance beyond initial expectations, making them spectroscopically complementary techniques. There are opportunities for these techniques to converge and complement each other, providing researchers with more insights into the chemical composition of materials at the nanoscale. AFM-IR and TERS have become crucial techniques for nanoscale chemical imaging and spectroscopy.

In another study by Lei Zhou and Kurouski, atomic force microscopy-infrared spectroscopy (AFM-IR) was used to examine the structure of individual α-synuclein oligomers at different stages of protein aggregation (3). The aggregation of α-synuclein protein is strongly linked to the development of Parkinson’s disease (PD). However, the structure and morphology of toxic intermediate oligomers formed during the aggregation process are not fully understood. The use of AFM-IR in this study allowed for the examination of individual oligomers at the nanoscale, which is critical for understanding the aggregation process and its relationship to protein structure.

Aberrant aggregation of α-synuclein protein is a hallmark of PD and other neurodegenerative disorders. The accumulation of α-synuclein in the form of intracellular inclusions, called Lewy bodies, is a pathological feature of PD. These inclusions are highly toxic to neurons, leading to neuronal dysfunction and degeneration. Additionally, soluble oligomeric forms of α-synuclein have been found to be highly neurotoxic and capable of inducing the formation of Lewy bodies. Therefore, understanding the structure and properties of α-synuclein oligomers and its role in the pathogenesis of PD is crucial for the development of effective diagnostic and therapeutic strategies.

The results of Zhou and Kurouski’s study showed that the morphology and structure of the oligomers changed with the progression of the aggregation process. The IR spectra of individual oligomers revealed structural rearrangements necessary for oligomers to propagate into fibrils with parallel-β-sheet secondary structure. This study provides insights into the structural organization of α-synuclein oligomers, which is essential to understand its toxicity. Moreover, the findings may contribute to the development of approaches for oligomer detection and pre-symptomatic diagnosis of PD.

Amyloid fibrils are associated with various neurodegenerative diseases and are of significant interest in structural biology. While conventional methods have provided insights into the morphology and core structure of fibrils, characterizing the surface has been challenging.

The formation of amyloid fibrils is a complex process that is associated with various neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as prion diseases. The accumulation of amyloid fibrils is believed to contribute to the degeneration of neurons and cognitive decline in these diseases. Understanding the structure and formation mechanism of amyloid fibrils is therefore crucial for developing effective treatments for these diseases. Raman spectroscopy has been extensively used to investigate protein aggregation and amyloid fibril formation due to its ability to reveal changes in secondary and tertiary structures at all stages of fibrillation. When combined with atomic force and scanning electron microscopies, 

Raman spectroscopy becomes a powerful approach for investigating the structural organization of amyloid fibril polymorphs, which can provide valuable insights into its formation and potential therapeutic targets.

, Kurouski and colleagues used tip-enhanced Raman spectroscopy (TERS) to probe the surface structure of insulin fibrils (4). The study demonstrated that TERS can provide high-resolution insights into the secondary structure and amino acid residue composition of the fibril surface. The insulin fibril surface was found to be strongly heterogeneous, with clusters exhibiting different protein conformations. More than 30% of the surface was dominated by β-sheet secondary structure, supporting Dobson’s model of amyloid fibrils. The study also revealed the distribution of various amino acids on the fibril surface and specific surface secondary structure elements. Cysteine and aromatic amino acids such as phenylalanine and tyrosine were found to be enriched in β-sheet areas, whereas proline was only found in α-helical and unordered protein clusters. The study also showed that carboxyl, amino, and imino groups were nearly equally distributed over β-sheet and α-helix/unordered regions.

, Kurouski and coauthors explore the applications of Raman spectroscopy in the structural characterization of amyloidogenic proteins, prefibrillar oligomers, and mature fibrils (5). The review summarizes various techniques that have been used in conjunction with Raman spectroscopy to provide a comprehensive understanding of the structure and formation mechanism of amyloid fibrils. The article highlights that Raman spectroscopy is a unique, label-free and nondestructive technique that can reveal the structural changes of amyloid fibrils during the fibrillation process. Amyloid fibrils are protein aggregates that form when proteins misfold and aggregate together in a β-sheet structure.

Plant pathogens are microorganisms that can infect and cause diseases in plants, leading to significant yield losses and reduced crop quality. Maize kernels, which are important sources of food and feed, are particularly susceptible to various pathogens, such as fungi and bacteria. These pathogens can cause symptoms such as discoloration, mold growth, and reduced kernel size, which negatively impact both yield and quality. Early detection and identification of these pathogens are critical for effective disease management and prevention. In this context, nondestructive and noninvasive techniques such as Raman spectroscopy are promising tools for rapid and accurate diagnosis, as demonstrated in the study published in 

In this study, researchers aimed to develop a rapid and nondestructive method for detecting and identifying plant pathogens on maize kernels, which is crucial for improving crop yield. They used a hand-held Raman spectrometer along with chemometric analysis to distinguish between healthy and diseased maize kernels and different diseases with 100% accuracy. Raman spectroscopy is a noninvasive and nondestructive technique that provides information about the chemical structure of a specimen. The sample-agnostic and portable nature of the analysis suggests that it could be retooled for other crops and conducted autonomously. This approach offers advantages over traditional pathogen assaying methods such as polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA), which are time-consuming and destructive to the sample. The study demonstrated the feasibility of using Raman spectroscopy for pathogen detection and identification, which could potentially have a significant impact on crop management and food security. The use of handheld Raman spectrometers allows for the implementation of this technique in the field, enabling rapid and accurate diagnosis of plant diseases, and facilitating prompt management decisions that can help protect crops from further damage.

Kurouski’s contributions to the field of molecular spectroscopy have been both significant and varied. His work in the area of Raman spectroscopy has led to the development of new, noninvasive methods for analyzing plant health and has demonstrated the potential of this technique for use in agriculture. Additionally, his work in the area of nano-Raman-nano-IR imaging has shed new insights into the structure of amyloid oligomers and protein aggregates that are linked to neurodegenerative diseases. His work in the area of plasmon-driven catalysis has elucidated the underlying physical causes of unique reactivity and selectivity in bimetallic nanostructures.

Kurouski’s accomplishments have not gone unnoticed, as evidenced by his numerous awards and honors, including the 2023 Emerging Leader in Molecular Spectroscopy Award from 

magazine. His publication record, which includes over 140 papers with more than 4,000 citations, and his extensive record of conference presentations demonstrate the impact of his research on the field of molecular spectroscopy.

Looking to the future, it seems likely that Kurouski will continue to make significant contributions to the field of molecular spectroscopy. His work has already demonstrated the potential of Raman spectroscopy for use in agriculture and for diagnostics of viral particles. As his research continues to progress, it seems likely that his work will continue to open new doors in this exciting and important field.

(1) Kurouski, D.; Postiglione, T.; Deckert-Gaudig, T.; Deckert, V.; Lednev, I. K. Amide I vibrational mode suppression in surface (SERS) and tip (TERS) enhanced Raman spectra of protein specimens. 

 (6), 1665–1673. DOI: 10.1039/C2AN36478F

(2) Kurouski, D.; Dazzi, A.; Zenobi, R.; Centrone, A. Infrared and Raman chemical imaging and spectroscopy at the nanoscale. 

 (11), 3315–3347. DOI: 10.1039/C8CS00916C

(3) Zhou, L.; Kurouski, D. Structural Characterization of Individual α-Synuclein Oligomers Formed at Different Stages of Protein Aggregation by Atomic Force Microscopy-Infrared Spectroscopy. 

 (10), 6806–6810. DOI: 10.1021/acs.analchem.0c00593

(4) Kurouski D.; Deckert-Gaudig T.; Deckert V.; Lednev I.K. Structure and Composition of Insulin Fibril Surfaces Probed by TERS. 

(32), 13323–13329. DOI: 10.1021/ja303263y

(5) Kurouski, D.; Van Duyne, R. P.; Lednev, I. K. Exploring the structure and formation mechanism of amyloid fibrils by Raman spectroscopy: a review. 

 (15), 4967–4980. DOI: 10.1039/C5AN00342C

(6) Farber, C.; Kurouski, D. Detection and Identification of Plant Pathogens on Maize Kernels with a Hand-Held Raman Spectrometer. 

 (5), 3009–3012. DOI: 10.1021/acs.analchem.8b00222

 serves on the Editorial Advisory Board of 

. He is the co-host of the Analytically Speaking podcast and has published multiple reference text volumes, including the three-volume 

Academic Press Handbook of Organic Compounds

The Concise Handbook of Analytical Spectroscopy

Practical Guide and Spectral Atlas for Interpretive Near-Infrared Spectroscopy

Dmitry Kurouski is this year’s recipient of the 2023 Emerging Leader in Molecular Spectroscopy Award for his work in using Raman spectroscopy for noninvasive, nondestructive analyses of biological materials.

 describes the development of a new fluorescent sensor for determining tetracycline (TC). The sensor uses a fluorescent probe comprised of adenine thymine (AT)-rich single-stranded DNA (ssDNA) templated copper nanoclusters (CuNCs) and could be as effective as fluorescence.

The fluorescent ssDNA-CuNCs were synthesized through a one-step method, utilizing AT-rich ssDNA as templates and ascorbic acid as reducing agents. One of the most notable features of the developed ssDNA-CuNCs is the potent fluorescence, characterized by a substantial Stokes shift of 240 nm and consistent fluorescence emission (1). This inherent stability of fluorescence was crucial for the researchers to obtain accurate and reliable measurements.

The researchers harnessed the spectral properties of ssDNA-CuNCs to design a responsive detection mechanism for TC (1). When tetracycline molecules were introduced, they interacted with the ssDNA-CuNCs, causing a noticeable reduction in fluorescent intensity. This phenomenon is known as the inner filter effect (1). This arises because of the spectral overlap between ssDNA-CuNCs and TC. The team optimized the experimental conditions to achieve the most sensitive detection.

To showcase the real-world application of this sensor, the researchers tested its performance on milk samples spiked with different concentrations of tetracycline (1). The results demonstrated the sensor's applicability and reliability in complex matrices, making it a promising tool for food safety and quality control (1).

Rapid and accurate detection methods for food safety are important to human health, and the development of this sensor could be another useful tool in food and beverage analysis (1). The development of this sensor marks a step in biosensing technology. As industries strive for more reliable and efficient methods of ensuring product safety and quality, this tool offers a glimpse into a future where advanced sensors are used to detect and quantify substances.

(1) Wu, N.-N.; Chen, L.-G.; Wang, H.-B. A Sensitive Fluorescence Sensor for Tetracycline Determination Based on Adenine Thymine-Rich Single-Stranded DNA-Templated Copper Nanoclusters. 

This article was written with the help of artificial intelligence and has been edited to ensure accuracy and clarity. You can read more about our

In a recent study, a new fluorescent sensor was used to determine tetracycline in milk.

Detecting Tetracycline In Milk Using Fluorescent Sensors

Fourier transform infrared (FT-IR) spectroscopy has attracted great attention in the field of disease diagnostics for many years. Yet, the clinical translation of FT-IR spectroscopy has been rather slow because of several bottlenecks. This work explores the potential of aluminum foil-assisted attenuated total reflectance-FT-IR (ATR-FT-IR) spectroscopy, which is a simple and economical FT-IR sampling technique, to detect acute kidney injury (AKI) induced by gentamicin in a rat model. It was found that partial least squares-discriminant analysis (PLS-DA) could successfully discriminate between the AKI status and the healthy status with the plasma samples of the rats. An in-depth discussion on the advantages of aluminum-foil-assisted ATR-FT-IR spectroscopy in FT-IR-based disease diagnostics is also provided.

Fourier transform infrared (FT-IR) spectroscopy is a rapid, non-invasive, and label-free analytical technology and has attracted great attention in the field of disease diagnostics for many years (1–6). As a vibrational spectroscopic tool, FT-IR spectroscopy has the capability to detect the vibrations of all molecules, such as proteins, nucleic acids, lipids, and carbohydrates in a clinical specimen. Thus, FT-IR spectroscopy can provide a comprehensive omnic picture of the tested sample. Owing to its sensitivity to the compositional and structural changes inside the clinical specimen,
FT-IR-based diagnostics can provide a spectral biomarker for diseases. The spectral biomarker can be a single spectral band or multiple spectral bands demonstrating the disease characteristics, or the whole spectral fingerprint can be used to discriminate the different health status when enabled using chemometric analysis.

To date, FT-IR-based diagnostics has been applied to a variety of clinical samples, including tissues (7–9), plasma and serum (10–16), and body fluids (17–20). Many proof-of-principle studies have clearly demonstrated the potential of FT-IR-based diagnostics for human diseases in clinical translation. Some startup companies aiming at translation to clinical applications were also established (2).

Aluminum-foil-assisted ATR-FT-IR spectroscopy was an FT-IR sampling approach developed by Martin and coworkers in 2016 (21). When implementing this approach, kitchen-grade aluminum foil may be used as the solid substrate for sample deposition. After a simple drying step, the deposited sample is pressed against the ATR crystal for FT-IR measurement. This sampling approach avoids the direct use of the ATR crystal or other expensive substrates for sample deposition during ATR-FT-IR measurement. It could significantly reduce the sampling cost and simplify the sampling procedure in clinical applications. The aluminum-foil-assisted ATR-FT-IR approach has now emerged as an attractive sampling approach for FT-IR-based disease diagnostics (20, 22–25). For example, one demonstration was performed very recently by Nascimento et al. where they used this approach to detect Covid-19 within saliva samples. By combining chemometric analysis with the spectra, they reported reasonable accuracy and sensitivity for Covid-19 diagnostics (20). This work aims to further tackle the potential of the aluminum-foil-assisted ATR-FT-IR approach in disease diagnostics by using this technique to detect acute kidney injury (AKI) status induced by gentamicin in a rat model. We hope that this exploratory work is beneficial for the future development of FT-IR-based disease diagnostics.

 = 3) were purchased from Vital River Lab Animal Technology Co., Ltd (Beijing, China). The rats were housed with free access to food and water under constant temperature, humidity, and lighting (12 h light per day). The animal experiment was carried out strictly according to the ethics guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee of Tianjin Tasly Institute, Tianjin, China. The rats were dosed with gentamicin (160 mg/kg) in saline to establish an AKI rat model. The dose of gentamicin used in this work was referred to as that used in a previous publication (26,27). These rats were injected with gentamicin for three consecutive days; that is, on day one, day two, and day three. The gentamicin sulfate injection solution was obtained from CSPC Pharmaceutical Group Co., Ltd. (Shijiazhuang, China). Sodium chloride (NaCl) was in analytical grade and ordered from a local vendor.

Sample Preparations for ATR-FT-IR Measurements

The whole blood samples from the rats were collected through retro-orbital blood sampling. The samples taken on day one before gentamicin injections were from the healthy rats, and they were used as the control group in the chemometric analysis of plasma samples; the samples taken on day two and day three before gentamicin injections and on day four were from the rats affected by gentamicin (from the AKI rats), and they were used as the AKI group in the chemometric analysis of plasma samples. The whole blood sample was centrifuged at 3000 r/min for 10 min and the supernatant plasma was collected and stored at -80 °C for future ATR-FT-IR investigation. To prepare the plasma sample for aluminum-foil-assisted ATR-FT-IR measurement, a large piece of kitchen-grade aluminum foil was cleaned with alcohol and water and then air-dried. The large piece of aluminum foil was cut into small pieces with a size of 1 x 1 cm. The plasma sample was first thawed under ambient conditions and 10 µL of the sample was pipetted onto the small piece of aluminum foil. The aluminum foil with sample deposit was placed in an oven at 37 °C for 2 hr. The sample formed a dry film on the surface of the aluminum foil. The dry plasma sample film was used for ATR-FT-IR measurement.

Aluminum-Foil-Assisted ATR-FT-IR Measurement

The details of aluminum-foil-assisted ATR-FT-IR measurement are shown in Figure 1 Measurements were made using a Bruker Vertex 70 FT-IR spectrometer (Ettlingen, Germany) equipped with a DLaTGS detector. A Pike Technologies single-reflection ATR sampling accessory (Madison, Wisconsin) was employed. This ATR sampling accessory contained a diamond ATR crystal. As shown in Figure 1, when performing the measurement, the surface of aluminum foil having plasma deposition was pressed against the ATR diamond crystal surface with a manual pressure clamp from Pike Technologies. The high-pressure of the pressing device would ensure a close contact between the sample and the diamond crystal. For the FT-IR measurements, 4 cm

 resolution and 32 scans were chosen. In this work, each plasma sample was used to prepare five replicate samples on five different pieces of aluminum foil. For each piece of aluminum foil sample, 12 FT-IR spectra were taken by sampling at different points on the foil. The total number of the collected FT-IR spectra for each group (that is, the control group or the AKI group) is approximately 180.

FIGURE 1: Schematic illustration of aluminum-foil-assisted ATR-FT-IR set-up. (a) aluminum foil with sample deposition, (b) ATR element, and (c) pressure device.

Partial least squares discriminant analysis (PLS-DA) was performed using Unscrambler software (version 10.5) (Camo Analytics). The second derivative FT-IR spectra in the 1750–900 cm

 region were used for chemometric analysis. Before chemometric analysis, the raw absorption spectra were subjected to data pre-treatments including smoothing and normalization.

The aim of this work was to examine whether the aluminum-foil-assisted ATR-FT-IR spectroscopy could differentiate the rats in the AKI group from the rats in the control group. The FT-IR spectra in the spectral region of 4000–900 cm

A recent study used aluminum foil-assisted ATR-FT-IR spectroscopy to detect acute kidney injury (AKI) in a rat model using plasma samples. The results 
show how ATR-FT-IR could be used to study more types of clinical samples in the future.

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