CC BY
126
MOLECULAR ORIGIN OF SURFACE-ENHANCED RAMAN SPECTRA OF E. COLI SUSPENSIONS EXCITED AT 532 AND 785 NM USING SILVER NANOPARTICLE SOLS AS SERS SUBSTRATES
Durovich EA1, Evtushenko EG1,2 H, Senko OV1, Stepanov NA1, Efremenko EN1, Eremenko AV2, Kurochkin IN1,2
1 Faculty of Chemistry, Lomonosov Moscow State University, Moscow
2 Emanuel Institute of Biochemical Physics of RAS, Moscow
Research into the molecular origin of surface-enhanced Raman spectra (SERS) of bacteria is a crucial step in assessing the future of SERS-based discrimination and identification of bacteria in clinical analysis, food quality control, etc. Previous studies have revealed that at 785 nm excitation wavelength SERS of bacterial cells placed on a solid surface functionalized with in-situ grown aggregated gold nanoparticles covered with SiO2 originate from a mixture of 6 purine derivatives (adenine, guanine, AMP, hypoxanthine, xanthine, and uric acid) that are released by the cells into the medium. The aim of the present work was to investigate whether such interpretation is possible with a different class of SERS substrates: silver nanoparticle sols at excitation wavelengths of 785 and 532 nm. The suspension of the Escherichia coli DH5a strain was used as a model bacterium. Sols of silver nanoparticles were obtained by reducing silver nitrate in the presence of alkaline hydroxylamine hydrochloride. Number-weighted mean hydrodynamic diameter of the particles was 43±2 nm. We confirm that at both excitation wavelengths the spectra can be best described as a superposition of 4 purine derivatives: adenine, guanine, hypoxanthine, and xanthine. Importantly, we have discovered that 1) the spectra of the purine mixture are characteristic of viable cells only; 2) due to the variations in the concentrations of purine metabolites released by the cells into the surrounding medium the spectra of a bacterial strain can vary significantly when a silver nanoparticle sol is used as a SERS substrate.
Keywords: SERS of bacteria, E. coli, silver nanoparticles, purines
[х] Correspondence should be addressed: Evgeniy G. Evtushenko
Leninskie gory 1, bl. 3, Moscow, 119991; evtushenko@enzyme.chem.msu.ru
Received: 15.08.2018 Accepted: 09.09.2018
DOI: 10.24075/brsmu.2018.088
МОЛЕКУЛЯРНАЯ ПРИРОДА ГКР-СПЕКТРОВ СУСПЕНЗИИ E. COLI ПРИ ДЛИНАХ ВОЛН ВОЗБУЖДЕНИЯ 532 И 785 НМ С ИСПОЛЬЗОВАНИЕМ ЗОЛЕЙ НАНОЧАСТИЦ СЕРЕБРА В КАЧЕСТВЕ ГКР-СУБСТРАТОВ
Е. А. Дурович1, Е. Г. Евтушенко1,2 О. В. Сенько1, Н. А. Степанов1, Е. Н. Ефременко1, А. В. Еременко2, И. Н. Курочкин1*2
1 Химический факультет, Московский государственный университет имени М. В. Ломоносова, Москва
2 Институт биохимической физики имени Н. М. Эмануэля РАН, Москва
Вопрос о молекулярной природе спектров гигантского комбинационного рассеяния (ГКР) бактерий является ключевым для оценки перспектив их дискриминации и идентификации данным методом в целях клинической диагностики, обеспечения безопасности пищевых продуктов и др. Ранее было показано, что при использовании в качестве ГКР-субстрата агрегированных и покрытых слоем SiO2 золотых наночастиц на твердой поверхности источником спектра при длине волны возбуждения 785 нм является смесь шести пуриновых производных (аденина, гуанина, АМФ, гипоксантина, ксантина и мочевой кислоты), выделяемая клетками в раствор. Целью настоящей работы было показать применимость данной интерпретации спектров на примере суспензии клеток Escherichia coli штамма DH5a для другого класса ГКР-субстратов — золей наночастиц серебра при длинах волн возбуждения 785 и 532 нм. Золи получали восстановлением нитрата серебра хлоридом гидроксиламина в щелочной среде, среднечисловой размер частиц составил 43 ± 2 нм. Выявлены две важные особенности: во-первых, спектр пуриновых метаболитов регистрируется только при наличии живых клеток; во-вторых, при использовании золей наночастиц серебра в качестве ГКР-субстрата спектрам даже одного и того же штамма присуща значительная вариативность вследствие изменения соотношения концентраций пуриновых метаболитов, выделяемых клетками в раствор.
Ключевые слова: ГКР-спектры бактерий, E. coli, наночастицы серебра, пуриновые производные
1X1 Для корреспонденции: Евгений Геннадиевич Евтушенко
Ленинские горы, д. 1, стр. 3, г Москва, 119991; evtushenko@enzyme.chem.msu.ru
Статья получена: 15.08.2018 Статья принята к печати: 09.09.2018
DOI: 10.24075/vrgmu.2018.088
Surface-enhanced Raman spectroscopy (SERS) is an optical technique that relies on the amplification of the weak Raman signal emitted by a molecule located in close proximity to a metal surface with nanoscale roughness. Dating back a few decades, SERS still has not lost its appeal as a powerful detection technique. It is rapid and simple in instrumentation;
it can be optimized to achieve a very high sensitivity and measure multiple analytes. SERS can also be employed for local analysis. Metal nanostructures used for signal enhancement are referred to as SERS substrates and fall into two major categories: nanostructures on solid supports and colloidal sols of metal nanoparticles.
SER spectra of bacteria were first recorded at a 514.5 nm laser excitation wavelength (EW) from Escherichia coli and Bacillus megaterium [1]. It was soon discovered that excitation at 488 or 514.5 nm results in almost identical SER spectra from gram-positive and gram-negative bacteria, as well as their isolated cell membranes: all acquired spectra originated from reduced or oxidized riboflavin (RF) [2-4]. The RF extinction band overlaps with 488 and 514.5 nm EWs, which induces resonant enhancement of the RF spectrum [3]. RF is a component of cofactors of redox enzymes and electron transport proteins found in cell membranes. If a bacterial cell is applied onto the surface of a solid SERS substrate or, alternatively, metal nanoparticles are synthesized or absorbed onto the cell, RF will come to occur in close proximity to the metal surface [3, 4]. From a bioanalytical standpoint, it means that laser sources with short wavelengths of 488 and 514.5 nm cannot be used for the identification of or discrimination between different bacteria.
At the same time, bacterial cells excited at long incident EW (785 nm) have SER spectra that do not contain the bands characteristic of riboflavin. Moreover, SER spectra vary between bacterial species and sometimes strains, not to mention intact and inactivated samples of the same strain [5-7]. This inspired a hypothesis in the early 21st century about the feasibility of SERS for the identification of bacterial species and strains excited at 785 nm wavelength. If adopted, this approach would have sped up pathogen detection in patients' samples, food products, and environmental objects. However, the molecular origin of bacterial SER spectra at long EWs was vague. According to a proposed hypothesis, such spectra could originate from the molecules localized in the bacterial glycocalyx (a slime layer or a capsule) or bacterial envelope (a cell wall or a membrane), as was the case with short wavelength lasers. Some authors speculated that the spectra might originate from N-acetyl-D-glucosamine [6], amino acid residues, peptides, protein prosthetic groups, phospholipids, metabolites (such as glucose or acetoacetic acid), or DNA and its constituents (guanine and adenine) [5, 7-13]. However, the proposed hypotheses lacked substance: they interpreted the origin of individual bands only ignoring the full spectra. As a result, this area of science was long dominated by a formal mathematical approach that combined the method of principal component analysis (PCA) used to reduce the dimensionality of experimental data and discriminant or cluster analysis aimed to prove the feasibility of discrimination between bacterial genera, species and strains based on their SER spectra [6, 12-15].
But then a study published in 2016 demonstrated convincingly that it was 6 purine metabolites released into the medium by the bacterial cell that were the source of SER spectra at 785 nm EW for 10 investigated samples [16]. The purine derivatives included adenine, guanine, adenosine monophosphate, hypoxanthine, xanthine, and uric acid. The suggested interpretation imposes a dramatic limitation on the use of bacterial SER spectra for the identification/ discrimination of pathogens because the differences between their spectra are caused by only 6 secreted purine derivatives and not the whole diversity of molecules on the cell surface. For example, a hypoxanthine-free E. coli mutant with a silent adenosine deaminase gene was closer in its SER spectrum to Staphylococcus aureus than to the parent strain. The EW used in that experiment was 785 nm. Aggregated gold nanoparticles grown on a solid surface and coated with a thin silica layer were used as a SERS substrate. The aim of our study was to verify the authors' conclusions using a principally different type of SERS substrates (silver nanoparticle sols) and to investigate
the molecular origin of bacterial SER spectra at 532 nm EW lying between riboflavin-dominated (488 and 514.5 nm) and infrared (785 and 1,064 nm) spectral regions.
METHODS
E. coli DH5a (Thermo Fisher Scientific; USA) was used as a model strain. The cells were cultivated in a liquid culture medium consisting of 10.0 g/l tryptone (Difco; USA), 5.0 g/l yeast extract (Difco; USA) and 10.0 g/l chemically pure NaCl (pH 6.8) (Chimmed; Russia) at 37 °C for 14-16 h until the stationary phase was reached. According to the literature [17], the cultured cells should be washed thoroughly to remove the residual components of the culture medium. Bearing that in mind, we applied the following protocol. Briefly, the cells were pelleted in the Beckman J-2-21 centrifuge (Beckman Coulter; USA) at 8,000 rpm for 7 min. The pellet was washed in an equivalent volume of 0.9% NaCl. The procedure was repeated twice. The obtained biomass was diluted with 0.9% NaCl taken at a volume sufficient for obtaining a suspension of 1 • 108 cells per ml. The final concentration was determined spectrophotometrically at 540 nm.
For the experiments with partially inactivated bacteria, the suspensions were placed into a water bath preheated to 70 °C or 90 °C and kept at this temperature for 1 h. The degree of inactivation was inferred from the concentration of intracellular ATP measured by the luciferase-luciferin assay using the reagent kit and calibration standards by Lumtek; Russia.
A sol of Ag nanoparticles (AgNPs) was used as SERS substrate. The sol was prepared by reducing silver nitrate with hydroxylamine hydrochloride in the presence of sodium hydroxide using AgNO3 (ASC reagent, > 99.0%; Sigma-Aldrich; USA), NH2OH^HCl (purified; Prime Chemicals Group; Russia), and NaOH (reagent grade; Mosreaktiv; Russia). Following the original protocol [18], the silver nitrate solution was poured into the alkaline hydroxylamine solution. The final concentrations of the reagents in the mixture were 1 mM AgNO3, 1.5 mM NH2OH^HCl, and 3 mM NaOH. Sols older than 3 days were not used in the experiment.
The absorption spectra of the synthesized nanoparticles were measured in a UV-visible region (300-750 nm) with the cuvette spectrophotometer UV-1800 (Shimadzu; Japan). The size and concentration of AgNPs were measured by nanoparticle tracking analysis (NTA) using the Nanosight LM10 HS-BF system (Nanosight Ltd; UK).
The SER spectra of both intact and inactivated bacteria were recorded on the day of sample preparation. Until then, the samples were stored at +4 °C. Immediately before the measurement, a sample aliquot was centrifuged twice at 3,700 rpm for 5 min in the Biofuge A centrifuge (Heraeus Sepatech; Germany) and washed in an equivalent volume of deionized water. The obtained cell suspension in water was mixed with the AgNP sol at a ratio of 1:1 and incubated for 1 min. Then, the NaCl solution taken at a final concentration of 40 mM was introduced into the mixture to stimulate particle aggregation and enhance the signal. An aliquot of this mixture (260 pl) was transferred into a well of an aluminum wellplate to minimize the background signal and improve heat dissipation. The spectra were measured in 3 to 4 replicates per sample; the samples were stirred by pipetting between measurements.
To study the changes in the SER spectra over time, 5 ml of the E. coli suspension were transferred to deionized water following the procedure described above. The obtained water suspension was stored at +4 °C and its aliquots were picked
to register SER spectra over the course of 4 h.The filtrate was prepared by filtering the E. coli water suspension slowly using a syringe filter SFNY030022S (Membrane Solutions; USA) with a diameter of 30 mm and a pore size of 0.22 pm.
At 785 nm EW, SER spectra were recorded using the innoRam BWS445(B)-785S spectrometer (BWTek; USA) with a 785 nm diode laser source and a *20 PL L 20/0.40 objective. The instrument was operated at a measuring range of 64-3,011 cm-1 and resolution of 4 cm-1. The spectra were
recorded using the incident beam power of 42 mW, 5 s signal accumulation time, and averaging over 20 repeated scans. At 532 nm EW, SER spectra were recorded using the iRaman BWS415-532S spectrometer (BWTek; USA) with a 532 nm diode laser source and a *20 PL L 20/0.40 objective. The instrument was operated at a measuring range of 174-4,001 cm-1 and resolution of 4 cm-1. The spectra were recorded using the incident beam power of 20 mW; 5 s signal accumulation time, and averaging over 20 repeated scans.
Table. The table shows all spectral bands observed In the SER spectra of E. coli Including the filtrates of cell suspensions and their assignment to the purine metabolites whose spectra were characterized in [16]. A — adenine, G — guanine, Hx — hypoxanthine, X — xanthine
785 nm 532 nm Assignment
Peak position, cm-1 Band frequency in the spectra, % Peak position, cm-1 Band frequency in the spectra, %
502-515 75 502-512 50 X, G
522-540 100 526 75 G
549-550 25 Hx, A
561-574 100 X, G, A
621-633 50 617-623 (sh) 100 Hx, A, G
653-667 100 648-651 100 G, X, A
680-683 38 X, A
724-735 100 721-728 100 A, Hx
780-792 63 770 25 Hx
788 25 A
838-842 38 833 25 Tyrosine (?)
848-850 50
867-883 63 875-878 25 G, X
925-930 38 Hx
958-966 100 952-955 100 X, G, Hx, A
1002-1008 88 1000-1006 100 Phenylalanine (?), A+Hx+X interaction (?)
1027-1033 25 1024-1027 100 G, A, Hx
1043-1045 75 G, X
1084-1096 50 1085-1095 100 Hx
1115-1130 38 1129-1140 100 X, G, A
1157-1160 25 1154 25 Hx
1175-1189 63 G, A
1213-1215 25 1215-1233 100 G, Hx, A
1245-1251 50 1242 25 X
1267-1276 25 1276 25 G, A
1310-1315 25 X, A
1324-1334 63 1322-1325 75 Hx
1330-1331 50 Hx
1341 25 А
1362-1380 63 1371-1379 100 G, X, Hx, A
1389-1390 13 1399 25 X, Hx
1444-1453 88 1444 25 Hx, G, A
1464-1473 75 1457-1468 100 Hx, G, X, A
1508 13 1506 25 A+Hx+X interaction (?)
1528-1534 25 1532-1538 100 G, Hx, К
1568-1578 63 1567-1575 50 G, A
1582-1591 25 1584-1595 25 X, Hx
1630-1721 (broad) 75 1646 25 G, A
1692-1698 100 X, Hx, G
The recorded spectra were processed in OPUS 7.0 (Bruker Optik GmbH; Germany). The data outside the 500-1,800 cm-1 range was discarded, and the baseline was subtracted using the Background correction tool. Smoothing was not applied to determine peak positions and intensity. However, SERS data for plots was smoothened using the Smooth tool with a frame width of 9 cm-1. The spectra were processed using vector normalization for a clear visual representation of qualitative differences. Normalization was not performed when the intensities of the spectra were compared.
RESULTS
The popular hydroxylamine technique for the synthesis of AgNP sols [18] is simple and reproducible; the sols it yields significantly enhance the spectra emitted by various analytes, including bacterial cells [19-21]. The AgNP sols we prepared were transparent, deep yellowish-brown in color and did not contain any precipitate. They had a broad and intense absorption band in the near UV-blue region corresponding to the localized surface plasmon resonance of AgNPs with a maximum at 407-409 nm and absorption at this wavelength ranging from 16.5 to 18 (this accounts for 30-fold dilution with deionized water). The number-weighted mean hydrodynamic diameter of the particles measured by nanoparticle tracking analysis was 43 ± 2 nm in three independent AgNP batches. The total particle concentration was (8.0 ± 1.7) • 1011 particles per ml. The synthesized AgNS sols aggregated in 40 mM NaCl did not have their own SER spectra at both EW except
for a broad low-intensity band contributed by aluminum (the material of the plate) in the region between 1,200 and 1,700 cm-1. This band can be totally subtracted during data processing.
The reproducibility of intact E. coli SER spectra at 785 nm EW was tested in a series of different experiment. First, we repeatedly measured the spectra of the same mixture of E. coli + AgNPs + NaCl. Second, we measured the spectra of different aliquots of bacterial sample using the same and different AgNP batches. Third, we measured the spectra of independently cultured and isolated E. coli applied onto one and the same AgNP substrate. Repeatedly measured bacterial samples demonstrated good repeatability (Fig. 1A). The SER spectra of independently cultured bacterial samples varied considerably (Fig. 1B). The most significant variations were observed in the following spectral regions: 508-532; 655; 730-734; 958; 1,450; 1,570-1,576 cm-1.
E. coli stored in water at +4 °C for 4 h (Fig. 1C) demonstrated a gradual increase in the total intensity of the SER spectrum over time accompanied by a change in the intensity ratio of its individual bands. For example, the ratio I730 / I655 = 1.2 remained constant at all time points, but the ratio I1325 / I655 monotonously declined from its initial value of 1.6 to 1.0 over the course of 4 h.
The spectra of the intact E. coli suspension in water were compared to its filtrate (0.22 pm) in order to locate the molecules giving rise to the SER spectra (Fig. 1D). Considering the slow dynamics, the spectra of aliquots of the initial bacterial suspension were recorded before and after filtration. All spectral
-Si
B
2 replicates for aliquot 1 2 replicates for aliquot 2
■ Batch 1
Batch 2
А*» А. л _Batch 3
C
0 min 60 min 140 min 240 min
D
Cell suspension before filtration Cell suspension after filtration Filtrate 0.22 |jm
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Raman shift (cm-1)
Fig. 1. SER spectra of E. coli suspensions at 785 nm excitation wavelength. A. Repeatability of measurements for one aliquot and one cell batch. B. Reproducibility of the spectra for different batches of cell suspensions. C. Dynamics of SER spectra over time observed in cells stored in water at +4 °C. D. Comparison of the spectra of the cell suspension and the filtrate (0.22 pm) of the same suspension. Vector normalization was applied to the spectra (A, B); the spectra (C, D) were not normalized to demonstrate the difference in their intensity. Ranges of spectral differences are shown in gray
Intact E coli cells
Cells inactivated at +70 °C for 1 h
Cells inactivated at +90 °C for 1 h
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Raman shift (cm-1)
Fig. 2. Changes in the SER spectra of E. coli suspensions (785 nm) following inactivation by heating. The spectra were not normalized to demonstrate the difference in their intensity
bands observed for the cell suspension were present in the spectra of the filtrate. Moreover, the total intensity of the filtrate spectrum was significantly higher.
The SER spectra of intact cells and those inactivated at 70 °C or 90 °C for 60 min were compared in an attempt to understand whether the observed SER spectra can indicate the presence of viable E. coli or whether they come from an inactivated bacterial biomass (Fig. 2). The residual concentration of intracellular ATP was also measured in all three sample types (intact bacteria and cells inactivated at 70 °C and 90 °C) as it is indicative of cell viability. The ATP concentrations were 1 • 10-9, 5.6 • 10-12 and 4.1 • 10-12 mol per 1 ml of cell suspension, respectively. On the whole, considerable variability was observed in the number and position of spectral bands. However, the total intensity of the spectrum tended to decrease. The spectra of inactivated bacteria (90 °C) contained only 4 very low-intensity bands characteristic of intact E. coli (730; 1,002; 1,325, and 1,450 cm-1) and two low-intensity bands of the amide III (1,230-1,270 cm-1) and amide I (1,640-1.680 cm-1) regions.
The reproducibility of the SER spectra of intact E. coli was also tested at 532 nm EW. In this case, the intensity of the spectra was twice as high as that observed at 785 nm, resulting in a higher number of informative spectral bands and a better accuracy in locating their position. Similar to 785 nm EW, at 532 nm the high repeatability of the spectra was observed for one and the same aliquot of one and the same bacterial sample
(Fig. 3A). But the SER spectra of independently cultured and isolated bacteria varied considerably (Fig. 3B).
DISCUSSION
Measurements conducted at EW 785 nm demonstrate that the SER spectra of E. coli stored in water are not determined by a single compound, but rather by a mixture of a few different components. This becomes clear when we look at the array of all recorded spectra that contains a fixed set of spectral bands (see the Table). The ratios of the mixture components slowly change over time when cells are stored in water (Fig. 1C) and differ significantly between independently cultured batches of intact cells (Fig. 1B). Comparison of the spectra of the intact bacterial preparation and its filtrate (Fig. 1D) shows that the components of the mixture do not originate from the cell surface but are present in the solution. Moreover, the cell itself can be seen as interfering with the recording of SER spectra, as it adsorbs particles on its surface. This is suggested by a significant increase in the total intensity of the filtrate spectrum in comparison with that of the cell suspension.
Inactivation of bacterial cells demonstrates that the mixture of the compounds in question bears connection to cell viability (Fig. 2) but is not a product of passive desorption from the surface of inactivated cells.
The Table features a list of spectral bands observed in all acquired SER spectra of intact E. coli, including the filtrates.
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Raman shift (cm-1)
Fig. 3. SER spectra of E. coli suspensions at 532 nm excitation wavelength A. Repeatability of measurements for one aliquot and one cell batch. B. Reproducibility of the spectra for different batches of cell suspensions. Vector normalization was applied to the spectra. Ranges of spectral differences are shown in gray
L < — Adenine - Guanine —■ Hypoxanthine - Xanthine i.„, ♦ ______
Intensity (a. u.) b A J W+iV/l ,1 huí i Adenine Guanine Hypoxanthine Xanthine llhl . ill, in, 11 , I'-Jfa.-,- .
4 J 1 i L^x. J , i i - Oxidized riboflavin - Reduced riboflavin 1 1 ll 1 X
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Raman shift (cm-1)
Fig. 4. Overlay of SER spectra corresponding to purine derivatives [16] and riboflavin [4, 24, 25] on the SER spectra of E. coli suspension A. Spectral bands of purine derivatives in the spectra of E. coli at 785 nm EW. Arrows mark uncharacterized low-intensity bands. B. Spectral bands of purine derivatives in the spectra of E. coli at 532 nm EW. C. Overlay of spectral bands of riboflavin on the spectra of E. coli at 532 nm EW. The width of riboflavin bands on the graph reflects the variability of their positions in literature sources
Upon analyzing the literature, we concluded that at 785 nm EW almost all spectral bands are a product of superposition of spectra originating from 4 purine derivatives (adenine, guanine, hypoxanthine, and xanthine). This conclusion is consistent with [16]. Besides, for every individual SER spectrum, the intensity and positions of bands correspond to such superposition as well. (Fig. 4A). Unlike the authors of [16] who exploited aggregated gold nanoparticles on a solid surface, we used sols of silver nanoparticles. Considering the possibility of slight variations in the relative intensity of the spectral bands associated with the use of different SER substrates and an increase in intensity following overlay of spectral bands of individual compounds, our description is quite accurate.
Only 3 low-intensity bands remain uncharacterized: 838842, 1,002-1,008 and 1,508 cm-1. On the one hand, there is a chance that low-intensity bands can be lost during digital conversion of the spectra from literature sources. On the other hands, it is possible that those bands have never been present in the spectra of individual purine derivatives. Then, their origin can be explained by two hypotheses. First, the bands can result from the interactions between the components of the mixture. For example, a SER spectrum of a mixture consisting of adenine, hypoxanthine and xanthine contains two bands (1,000 and 1,510 cm-1) absent in the individual spectra of its constituents [22]. Besides, the presence of 838-842 and 1,002 cm-1 bands can be explained by minor presence of tyrosine (the most intense spectral bands are 824; 847; 928; 1,046; 1,389 and 1,583 cm-1 [23]) and phenylalanine (the most intense spectral bands are 930; 1,002; 1,031; 1,394; and 1,602 cm-1 [23]).
The suggested origin of bacterial SER spectra explains a considerable variation in the position of peaks observed for some bands within E. coli spectra. Thus, the variability in the position of the peak of a broad multicomponent band ranging from 502 to 574 cm-1 can be explained by an overlap of the following bands: xanthine (508 cm-1), guanine (526 cm-1), hypoxanthine (550 cm-1), adenine (558 cm-1), and guanine (577 cm-1). For the band in the region between 653 and 667 cm-1, the contributing bands are 652 cm-1 (adenine), 657 cm-1 (xanthine) and 667 cm-1 (guanine); for the 724-735 cm-1 band, 725 cm-1 (hypoxanthine) and 734 cm-1 (adenine); for the broad double band with peaks at 1,444-1,452 cm-1 and 1,4641,473 cm-1, the contribution is made by guanine (1447 cm-1), adenine (1455 cm-1), hypoxanthine (1456 cm-1), guanine (1466 cm-1), and xanthine (1478 cm-1).
The SER spectra of E. coli are very similar at 785 and 532 nm EW (Fig. 4A, 4B) with regards to the position and intensity of some of their constituting bands (Fig. 1B, 3B). This finding encouraged us to describe the acquired SER spectra at EW
532 nm as representing a mixture of 4 purine metabolites (adenine, guanine, hypoxanthine, and xanthine) as well. The reference spectra characterized in [16] differ from the acquired E. coli spectra in the type of the SER substrate used and EW, resulting in slight shifts in band positions. Considering that, our description of the experimental SER spectra of E.coli at EW 532 nm can be characterized as satisfactory.
We also explored a possibility of ascribing the bands in the SER spectra of E. coli at 532 nm EW to reduced or oxidized riboflavin and FAD whose reference spectra were borrowed from some early works [4, 24, 25] (Fig. 4C). A few high and medium intensity bands of E. coli are absent in the SER spectra of RF including 650, 725-733, 955, and 1,365 cm-1. In turn, the SER spectra of E. coli either miss a number of RF bands or include the bands with a strongly different intensity: 528529, 834-839, 1,149-1,156, 1,279-1,289, 1,491-1,502, and 1,523-1,527 cm-1 for oxidized RF and 528, 1,251, 1,501 and 1,530 cm-1 for reduced RF. Nevertheless, the contribution of the RF to the E. coli spectra at 532 nm EW cannot be ruled out. It could additionally increase the intensity of a broad band in the region between 1,300 and 1,350 cm-1 in comparison with the mixture of purine derivatives. However, RF does not dominate the spectrum. This somewhat contradicts the early conclusions about its dominance in the spectra of Pseudomonas aeruginosa, Bacillus subtilis, and Geobacillus stearothermophilus at 532 nm [25]. Such discrepancy can be explained by the difference in the used bacteria species.To sum up, the SER spectra of E. coli at 532 nm EW can be best described as a superposition of the spectra of purine derivatives. Similarly to 785 EW, the variability of the spectra of bacterial samples from different batches at 532 nm EW results from the difference in the concentrations of these compounds released by the cells into the solution.
CONCLUSIONS
The SER spectra of E. coli excited at 785 and 532 nm originate from a mixture of purine derivatives released by the cells into the solution, given that a silver nanoparticle sol synthesized following the hydroxylamine technique is used as a SERS substrate. For both excitation wavelengths, the acquired spectra are best described as originating from adenine, guanine, hypoxanthine, and xanthine. Riboflavin may slightly contribute to the spectra excited at 532 nm. The acquired spectra are characteristic of viable bacteria cells only. Their variability results from the differences in the ratio of the contributing components. Such molecular origin of bacterial SER spectra imposes serious limitations on the use of SERS for bacterial identification and discrimination.
References
1. Efrima S, Bronk BV. Silver Colloids Impregnating or Coating Bacteria. J Phys Chem B. 1998; 102 (31): 5947-50.
2. Zeiri L, Bronk BV, Shabtai Y, Czege J, Efrima S. Silver metal induced surface enhanced Raman of bacteria. Colloids Surfaces A Physicochem Eng Asp. 2002; 208 (1): 357-62.
3. Picorel R, Lu T, Holt RE, Cotton TM, Seibert M. Surface-Enhanced Resonance Raman Scattering (SERRS) Spectroscopy of Bacterial Membranes: The Flavoproteins. In: Baltscheffsky M, editor. Current Research in Photosynthesis: Proceedings of the VIIIth International Conference on Photosynthesis; 1989 Aug 6-11; Stockholm, Sweden. Dordrecht: Springer Netherlands, 1990; p. 1867-70.
4. Zeiri L, Bronk BV, Shabtai Y, Eichler J, Efrima S. Surface-Enhanced Raman Spectroscopy as a Tool for Probing Specific Biochemical Components in Bacteria. Appl Spectrosc. 2004; 58 (1): 33-40.
5. Guzelian AA, Sylvia JM, Janni JA, Clauson SL, Spencer KM. SERS of whole-cell bacteria and trace levels of biological molecules. Proc. SPIE, Vibrational Spectroscopy-Based Sensor Systems. 2002; (4577): 183-92.
6. Jarvis RM, Goodacre R. Discrimination of Bacteria Using Surface-Enhanced Raman Spectroscopy. Anal Chem. 2004; 76 (1): 40-7.
7. Premasiri WR, Moir DT, Klempner MS, Krieger N, Jones G, Ziegler LD. Characterization of the Surface Enhanced Raman Scattering (SERS) of Bacteria. J Phys Chem B. 2005; 109 (1): 312-20.
8. Luo BS, Lin M. A Portable Raman System for the Identification of Foodborne Pathogenic Bacteria. J Rapid Methods Autom Microbiol. 2008; 16 (3): 238-55.
9. Kahraman M, Keseroglu K, Çulha M. On sample preparation for surface-enhanced Raman scattering (SERS) of bacteria and the source of spectral features of the spectra. Appl Spectrosc. 2011; 65 (5): 500-6.
10. Feng J, de la Fuente-Nünez C, Trimble MJ, Xu J, Hancock REW, Lu X. An in situ Raman spectroscopy-based microfluidic "lab-on-a-chip" platform for non-destructive and continuous characterization of Pseudomonas aeruginosa biofilms. Chem Commun. 2015; 51 (43): 8966-9.
11. Su L, Zhang P, Zheng D, Wang Y, Zhong R. Rapid detection of Escherichia coli and Salmonella typhimurium by surface-enhanced Raman scattering. Optoelectron Lett. 2015; 11 (2): 157-160.
12. Mosier-Boss AP. Review on SERS of Bacteria. Biosensors. 2017; 7 (4): 51-76.
13. Witkowska E, Korsak D, Kowalska A, Janeczek A, Kaminska A. Strain-level typing and identification of bacteria — a novel approach for SERS active plasmonic nanostructures. Anal Bioanal Chem. 2018; 410 (20): 5019-31.
14. Patel IS, Premasiri WR, Moir DT, Ziegler LD. Barcoding bacterial cells: a SERS-based methodology for pathogen identification. J Raman Spectrosc. 2008; 39 (11): 1660-72.
15. Sundaram J, Park B, Hinton A, Lawrence KC, Kwon Y. Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique. J Food Meas Charact. 2013; 7 (1): 1-12.
16. Premasiri WR, Lee JC, Sauer-Budge A, Théberge R, Costello CE, Ziegler LD. The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS. Anal Bioanal Chem. 2016; 408 (17): 4631-47.
17. Marotta NE, Bottomley LA. Surface-Enhanced Raman Scattering
of Bacterial Cell Culture Growth Media. Appl Spectrosc. 2010; 64 (6): 601-6.
18. Leopold N, Lendl B. A New Method for Fast Preparation of Highly Surface-Enhanced Raman Scattering (SERS) Active Silver Colloids at Room Temperature by Reduction of Silver Nitrate with Hydroxylamine Hydrochloride. J Phys Chem B. 2003; 107 (24): 5723-7.
19. Cañamares MV, Garcia-Ramos JV, Sanchez-Cortes S, Castillejo M, Oujja M. Comparative SERS effectiveness of silver nanoparticles prepared by different methods: A study of the enhancement factor and the interfacial properties. J Colloid Interface Sci. 2008; 326 (1): 103-9.
20. Knauer M, Ivleva NP, Niessner R, Haisch C. Optimized Surface-enhanced Raman Scattering (SERS) Colloids for the Characterization of Microorganisms. Anal Sci. 2010; 26 (7): 761-6.
21. Félix-Rivera H, González R, Rodríguez GDM, Primera-Pedrozo OM, Ríos-Velázquez C, Hernández-Rivera SP. Improving SERS Detection of Bacillus thuringiensis Using Silver Nanoparticles Reduced with Hydroxylamine and with Citrate Capped Borohydride. Int J Spectrosc. 2011; Article ID 989504.
22. Ranc V, Hruzikova J, Maitner K, Prucek R, Milde D, Kvítek L. Quantification of purine basis in their mixtures at femto-molar concentration levels using FT-SERS. J Raman Spectrosc. 2011; 43 (8): 971-6.
23. Kim SK, Kim MS, Suh SW. Surface-enhanced Raman scattering (SERS) of aromatic amino acids and their glycyl dipeptides in silver sol. J Raman Spectrosc. 1987; 18 (3): 171-5.
24. Kazanci M, Schulte JP, Douglas C, Fratzl P, Pink D, Smith-Palmer T. Tuning the Surface-Enhanced Raman Scattering Effect to Different Molecular Groups by Switching the Silver Colloid Solution pH. Appl Spectrosc. 2009; 63 (2): 214-3.
25. Smith-Palmer T, Douglas C, Fredericks P. Rationalizing the SER spectra of bacteria. Vib Spectrosc. 2010; 53 (1): 103-6.
Литература
1. Efrima S, Bronk BV. Silver Colloids Impregnating or Coating Bacteria. J Phys Chem B. 1998; 102 (31): 5947-50.
2. Zeiri L, Bronk BV, Shabtai Y, Czege J, Efrima S. Silver metal 11. induced surface enhanced Raman of bacteria. Colloids Surfaces
A Physicochem Eng Asp. 2002; 208 (1): 357-62.
3. Picorel R, Lu T, Holt RE, Cotton TM, Seibert M. Surface-Enhanced 12. Resonance Raman Scattering (SERRS) Spectroscopy of Bacterial Membranes: The Flavoproteins. In: Baltscheffsky M, editor. 13. Current Research in Photosynthesis: Proceedings of the VIIIth International Conference on Photosynthesis; 1989 Aug 6-11; Stockholm, Sweden. Dordrecht: Springer Netherlands, 1990; p. 1867-70. 14.
4. Zeiri L, Bronk BV, Shabtai Y, Eichler J, Efrima S. Surface-Enhanced Raman Spectroscopy as a Tool for Probing Specific Biochemical Components in Bacteria. Appl Spectrosc. 2004; 58 (1): 33-40. 15.
5. Guzelian AA, Sylvia JM, Janni JA, Clauson SL, Spencer KM. SERS of whole-cell bacteria and trace levels of biological molecules. Proc. SPIE, Vibrational Spectroscopy-Based Sensor Systems. 2002; (4577): 183-92. 16.
6. Jarvis RM, Goodacre R. Discrimination of Bacteria Using Surface-Enhanced Raman Spectroscopy. Anal Chem. 2004; 76 (1): 40-7.
7. Premasiri WR, Moir DT, Klempner MS, Krieger N, Jones G, Ziegler LD. Characterization of the Surface Enhanced Raman 17. Scattering (SERS) of Bacteria. J Phys Chem B. 2005; 109 (1): 312-20.
8. Luo BS, Lin M. A Portable Raman System for the Identification
of Foodborne Pathogenic Bacteria. J Rapid Methods Autom 18. Microbiol. 2008; 16 (3): 238-55.
9. Kahraman M, Keseroglu K, Qulha M. On sample preparation for surface-enhanced Raman scattering (SERS) of bacteria and the source of spectral features of the spectra. Appl Spectrosc. 2011;
65 (5): 500-6. 19.
10. Feng J, de la Fuente-Nünez C, Trimble MJ, Xu J, Hancock REW, Lu X. An in situ Raman spectroscopy-based microfluidic "lab-on-a-chip" platform for non-destructive and continuous
characterization of Pseudomonas aeruginosa biofilms. Chem Commun. 2015; 51 (43): 8966-9.
Su L, Zhang P, Zheng D, Wang Y, Zhong R. Rapid detection of Escherichia coli and Salmonella typhimurium by surface-enhanced Raman scattering. Optoelectron Lett. 2015; 11 (2): 157-160. Mosier-Boss AP. Review on SERS of Bacteria. Biosensors. 2017; 7 (4): 51-76.
Witkowska E, Korsak D, Kowalska A, Janeczek A, Kaminska A. Strain-level typing and identification of bacteria — a novel approach for SERS active plasmonic nanostructures. Anal Bioanal Chem. 2018; 410 (20): 5019-31.
Patel IS, Premasiri WR, Moir DT, Ziegler LD. Barcoding bacterial cells: a SERS-based methodology for pathogen identification. J Raman Spectrosc. 2008; 39 (11): 1660-72. Sundaram J, Park B, Hinton A, Lawrence KC, Kwon Y. Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique. J Food Meas Charact. 2013; 7 (1): 1-12.
Premasiri WR, Lee JC, Sauer-Budge A, Théberge R, Costello CE, Ziegler LD. The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS. Anal Bioanal Chem. 2016; 408 (17): 4631-47. Marotta NE, Bottomley LA. Surface-Enhanced Raman Scattering of Bacterial Cell Culture Growth Media. Appl Spectrosc. 2010; 64 (6): 601-6.
Leopold N, Lendl B. A New Method for Fast Preparation of Highly Surface-Enhanced Raman Scattering (SERS) Active Silver Colloids at Room Temperature by Reduction of Silver Nitrate with Hydroxylamine Hydrochloride. J Phys Chem B. 2003; 107 (24): 5723-7.
Cañamares MV, Garcia-Ramos JV, Sanchez-Cortes S, Castillejo M, Oujja M. Comparative SERS effectiveness of silver nanoparticles prepared by different methods: A study of the enhancement factor and the interfacial properties. J Colloid Interface Sci. 2008;
326 (1): 103-9.
20. Knauer M, Ivleva NP, Niessner R, Haisch C. Optimized Surface-enhanced Raman Scattering (SERS) Colloids for the Characterization of Microorganisms. Anal Sci. 2010; 26 (7): 761-6.
21. Félix-Rivera H, González R, Rodríguez GDM, Primera-Pedrozo OM, Ríos-Velázquez C, Hernández-Rivera SP. Improving SERS Detection of Bacillus thuringiensis Using Silver Nanoparticles Reduced with Hydroxylamine and with Citrate Capped Borohydride. Int J Spectrosc. 2011; Article ID 989504.
22. Ranc V, Hruzikova J, Maitner K, Prucek R, Milde D, Kvítek L. Quantification of purine basis in their mixtures at femto-molar
concentration levels using FT-SERS. J Raman Spectrosc. 2011; 43 (8): 971-6.
23. Kim SK, Kim MS, Suh SW. Surface-enhanced Raman scattering (SERS) of aromatic amino acids and their glycyl dipeptides in silver sol. J Raman Spectrosc. 1987; 18 (3): 171-5.
24. Kazanci M, Schulte JP, Douglas C, Fratzl P, Pink D, Smith-Palmer T. Tuning the Surface-Enhanced Raman Scattering Effect to Different Molecular Groups by Switching the Silver Colloid Solution pH. Appl Spectrosc. 2009; 63 (2): 214-3.
25. Smith-Palmer T, Douglas C, Fredericks P. Rationalizing the SER spectra of bacteria. Vib Spectrosc. 2010; 53 (1): 103-6.
