A-TEEM Molecular Fingerprinting

A-TEEM Spectroscopy

A-TEEM Molecular Fingerprinting Interview

What is A-TEEM Molecular Fingerprinting?

A-TEEM™ Molecular Fingerprinting is a new optical technique that is ideal for comprehensive component analysis in a variety QC/QA applications, from water to wine to pharmaceuticals and more. It is a simple, fast, “column free” spectroscopic technique that simultaneously measures the absorbance, transmission and fluorescence of samples in solution, and offers unique benefits over traditional analytical techniques, such as chromatography, mass spec, IR, etc.. Fundamentally, this technique is best applied to samples in solution that have fluorescing components, like an aromatic molecule, although it also detects non-fluorescing species via absorbance. The fluorescence EEM fingerprint technique has also been applied to solids and skin. Fluorescence is a highly sensitive technique compared to other spectroscopic techniques such as absorbance, FTIR and Raman, and the three-dimensional nature of the data provides for a better foundation for complex component analysis.

A-TEEM spectroscopy refers to the ability to simultaneously acquire Absorbance, Transmittance and a fluorescence Excitation Emission Matrix (A-TEEM) of a particular sample. HORIBA pioneered this technique with the patented Aqualog system, which combines A-TEEM spectroscopy with multichannel CCD detection to provide extremely fast results.

The A-TEEM technique requires no sample preparation, beyond possibly a simple dilution, and the cost and ease of use of an A-TEEM spectrometer allows it to be used both in the research lab as well as at-line or in-line in a production process. It’s a simpler, faster, and cheaper column-free chromatography technique for fluorescence components in solution. A-TEEM has taken fluorescence spectroscopy beyond the research lab and into the analytical industrial QC/QA environment.

HORIBA A-TEEM spectrometers can be used for fluorescence EEMs and for absorbance measurements for multi-component analysis and for transmission measurements for CIE colorimetry.

Absorbance
Absorbance of Anthracene

Absorbance of Anthracene

Transmission/Color
CIE chromaticity plot of various wines

CIE chromaticity plot of various wines

Fluorescence
A-TEEM 3D Fingerprint
Aqualog A-TEEM fingerprint of RPMi1 cell culture media

Aqualog A-TEEM fingerprint of RPMi1 cell culture media

The remarkable power of A-TEEM spectroscopy is derived from the fact that the three-dimensional fluorescence EEMs collected by the instrument are corrected for the inner filter effect. This means they are true and accurate representations of the molecules of interest over a much broader, and more useable, concentration range (typically up to ~2 absorbance units). Therefore, these EEMs allow for much more precise fingerprinting than is possible with an EEM collected from a traditional scanning fluorometer.

A-TEEM spectroscopy now brings the fluorescence technique into the true analytical market and it has demonstrated that it can, in some cases, replace traditional instruments, like an HPLC or mass spectrometer, as a simpler, faster and less expensive analytical tool.

HORIBA A-TEEM spectrometers are powered by Solo Predictor software from Eigenvector Research Incorporated. To access the true power of fluorescence A-TEEM spectroscopy, one needs to employ multivariate analysis methods (chemometrics) such as Principal Components Analysis (PCA), Classical Least Squared (CLS) method and Parallel Factor Analysis (PARAFAC), to perform component and concentration analysis of the three-dimensional data sets. Many sample measurements are used to create a model and then you can use chemometrics to get scores of each component in an individual sample.

Fluorescence is a highly sensitive technique and the three-dimensional A-TEEM fingerprint technique that allows us to distinguish subtle variations in molecular structure, due to changes in the excitation and emission characteristics.

A-TEEM fingerprints

Although these three-dimensional A-TEEM fingerprints look very similar, they are easily distinguished with chemometrics software, acquired with Aqualog.

A-TEEM versus Traditional EEM

Traditional scanning spectrofluorometers have been used to collect a molecular fingerprint, in the form of a fluorescence excitation emission matrix, or EEM. Sometimes also referred to as 3D Fluorescence, an EEM is a three-dimensional data set of fluorescence excitation wavelength versus fluorescence emission wavelength versus fluorescence intensity. With a scanning spectrofluorometer, this data set is acquired by sequentially scanning a series of emission spectra, at varying excitation wavelengths, and then reconstructing the resultant data set three dimensionally. This three-dimensional data set can be used with third party multivariate analysis software for component analysis, as is done with other analytical techniques such as FTIR, HPLC and MS. There are, in fact, many scientific papers published citing the use of scanning spectrofluorometers for fluorescence EEM component analysis in many disciplines including food sciences, water research and pharmaceuticals.

There are, however, two fundamental limitations of using a traditional scanning PMT fluorometer for EEM component studies. The first is that it takes a very long time to collect a single EEM with a scanning fluorometer. Depending on the brightness of the signal, and the wavelength range and resolution that is required, a single EEM experiment can take a scanning spectrofluorometer up to an hour to collect!

Another important limitation of scanning fluorometers is that the shape of the fluorescence EEM fingerprint itself can change with even subtle variations in sample concentration. If an instrument measures different EEM fingerprints for the same molecule at different concentrations, it really can’t be used for component analysis. For an EEM to be used as a true analytical technique, the shape of the spectra must be independent of concentration.

These two inherent limitations of a scanning spectrofluorometer have impacted the usability of the fluorescence EEM technique, and this has lead to the development by HORIBA of the A-TEEM technique.

HORIBA’s unique A-TEEM technique overcomes these two limitations. With CCD detection technology, HORIBA solves the serious speed limitations of scanning spectrofluorometers because with HORIBA technology, an entire fluorescence EEM can be acquired in mere seconds to minutes depending on the sample.

Three dimensional contour plot viewed at an angle of fluorescence EEM

Figure 1. Three dimensional contour plot viewed at an angle of a fluorescence EEM, with three dimensional axis for fluorescence excitation, emission and intensity.

Contour plot (top down view) of fluorescence EEM

Figure 2. Contour plot (top down view) of fluorescence EEM of fluorescein acquired in one second with Duetta.

HORIBA has also solved the problems associated with the fluorescence inner filter effect by taking advantage of the fact that the A-TEEM technique also collects absorbance of the same sample at the same time as the fluorescence, and uses the absorbance to correct EEMs for the inner filter effect (IFE).

HORIBA calls this technique A-TEEMTM, for Absorbance-Transmission Excitation Emission Matrix. By correcting for inner filter effects, the A-TEEM molecular fingerprint is a much more absolute representation of the true molecular fingerprint.  Therefore when when using third party multivariate chemometrics analysis software, the A-TEEM data provides much more robust component analysis than can be achieved with just a simple EEM from a scanning fluorometer.

Below is a good example to show how even a small concentration difference in a single molecule can have a significant effect on the shape of an EEM fingerprint, but with proper IFE correction an A-TEEM fingerprint remains the same.

Fluorescence EEMs of two concentrations of quinine sulfate

Figure 1. Fluorescence Excitation Emission Matrices of two concentrations of quinine sulfate in tonic water diluted in 0.1 M perchloric acid (aq.) with and without inner-filter effect corrections applied. Acquired with HORIBA Duetta.

Aqualog A-TEEM chemometrics analysis presented here are derived from Eigenvector Research Incorporated, Solo software.

 

A-TEEM Applications

A-TEEM for Water

A-TEEM for Pharma

A-TEEM for Wine

A-TEEM for Cosmetics

A-TEEM for Olive Oil

A-TEEM for Water

The A-TEEM technique was first introduced with the invention of the HORIBA Aqualog spectrometer in 2010. It was first designed as a much more accurate, sensitive and faster spectroscopy solution for water quality analysis, specifically for the study of chromophoric dissolved organic matter, also called CDOM. Dissolved organic matter includes amino acids, humic acids, fulvic acids, and other examples of decayed matter in natural water sources, or disinfection byproducts of water treatment processes. Prior to the introduction of the Aqualog, scientists were using traditional scanning spectrofluorometers for CDOM studies, which are very slow, and do not provide concentration independent fluorescence EEM fingerprints. Today, the HORIBA Aqualog is the gold standard for environmental water research, not just for CDOM but for many other species. Aqualog is also being used in water treatment plants around the world as a simple, fast early warning monitor and to save money by facilitating chemical treatment optimization.

Common fluorescent compounds in water include humic acids, fulvic acids, and amino acids

Common fluorescent compounds in water include humic acids, fulvic acids, and amino acids (waste water) which can be studied during various treatment processes, by PARAFAC analysis of the A-TEEM fingerprint. Acquired with Aqualog.

Most components of CDOM have broad overlapping fluorescence excitation and emission spectra in the UV and visible range. Many sample measurements are used to create a model and then and then chemometrics software is used to get scores of each component in an individual sample. The unique thing about a a fluorescence A-TEEM is that it can corrected for IFE. It can be used as a precise molecular fingerprint. Changes in the emission spectrum, the excitation spectrum, or both can be tracked very easily using this 3D fluorescence method for water analysis.

Aqualog A-TEEM at Drinking Water Treatment Plants

Aqualog is used at drinking water treatment plants to monitor and track key water parameters from three different sources: The raw water coming into the plant; the settled water inside the plant after sedimentation; and the finished water that goes into the drinking supply. Key parameters include the following:

  • THM Species
  • THMFP
  • DOC
  • SUVA
  • A254
  • Aromaticity Index

 

To learn more about how A-TEEM molecular fingerprinting can help you with your application, email us at a-teem.us@horiba.com

A-TEEM for Pharma

The A-TEEM technique, first introduced with the invention of the HORIBA Aqualog spectrometer in 2010, was designed as a much more accurate, sensitive and faster spectroscopy solution for water quality analysis.Now, Aqualog is being used in many new and very exciting industrial QC/QA applications. In the pharmaceutical industry there are two very exciting applications of Aqualog. The first is the use of A-TEEM fingerprinting upstream to monitor cell culture media. Another is the use of A-TEEM downstream to validate packaged vaccines. In both applications, the Aqualog provides a simple and fast optical method that can save a pharmaceutical company a tremendous amount of money compared to more traditional analytical methods. Most of the work Aqualog is doing in the pharmaceutical industry is protected under confidentiality agreements, however there is some information that can be shared about these applications.

Monitoring Cell Culture Media

With the rise of protein production using mammalian cell culture, it has become increasingly important to control the quality of the cell culture media for use in production processes. Cell culture media for bioreactors are usually prepared as an aqueous solution and provide everything a cell line needs for optimal growth as well as product yield and quality. Even subtle variations in composition could have a noticeable impact on the growth rate of the cell culture and its yield. Therefore the composition and quality of cell culture media in bioreactors must be tightly controlled in order to maintain an optimal bioreactor process.Thus, identifying and analyzing cell culture media is very important. As a result, the pharmaceutical industry has begun to turn to spectroscopic methods such as fluorescence for cell culture media analysis due to the speed of testing, minimal sample handling requirements, and relatively lower cost when compared to mass spectrometry and chromatography.

The composition and quality of cell culture media in bioreactors must be tightly controlled in order to maintain an optimal bioreactor process. As a result, methods of identifying and analyzing the quality of cell culture media have become an important focus in this field.

Common A-TEEM Measurements in Cell Media

Fluorescence Compounds

Absorbing Compounds

Color

Amino Acids

Nucleotides

 

Tryptophan

Proteins

 

Phenylalanine

Biomass

 

Tyrosin

 

 

NAD(P)H

 

 

FAD/FMN

 

 

Pyruvate

 

 
A-TEEM Benefits for Cell-Media QC/QA
  • A-TEEM provides comprehensive molecular fingerprints in seconds to minutes
  • A-TEEM requires no pumps, columns or solvents, and minimal sample preparation
  • Information rich and immediately compatible with a range of multivariate tools and unique multi-block capabilities
  • Ideal for component regression (confirm stoichiometry)
  • Ideal for media classification
  • Discrimination of component level changes
    • Light xxposure
    • Oxidation
    • Cell metabolism

Recently, it was shown that A-TEEM, along with the aid of certain chemometric methods (PARAFAC, PCA) provides a fast, effective, and inexpensive solution to identify and assess the quality of cell culture media.

 Examples of cell culture media molecular fingerprint A-TEEMs

Figure 1: Examples of cell culture media molecular fingerprint A-TEEMs. The media shown are from the samples DMEM1 (left), HAMSF12 (middle), and RPMI1 (right). Data acquired with Aqualog.

Unsupervised Classifications of Major Media Types and Variations

Even different modifications of the same media type can be discriminated using the Aqualog A-TEEM spectrometer. This figure shows a three component score plot that was generated with a Principal Component Analysis (PCA) using all data from three different DMEM samples. Data acquired on Aqualog, PCA analysis performed with Eigenvector Solo software.

Vaccine Validation

Instead of a potency test, the HORIBA Aqualog can be used to identify different vaccines based on subtle molecular variations obtained with a simple optical analysis. Together with a third-party CFR compliant software wrap, Aqualog can be applied to regulatory end point validation.

Insulin Structure and Stability

Insulin is a protein hormone, produced by the pancreas and is necessary for basic metabolic processes. It consists of intrinsically fluorescent amino acids. The different types of commercial insulin therapeutics generally fall into two categories: Short-acting and long-acting insulin. The difference between some short-acting and long-acting insulin is, in some cases, only one, two or three amino acids in the protein sequence, yet the A-TEEM molecular fingerprint can distinguish such subtle variations. The small sequence changes cause differences in the insulin protein aggregation behavior and local solvent environment of the fluorescent residues, and this can be this can be monitored using A-TEEM fingerprints.

Source: “A-TEEM™, a new molecular fingerprinting technique: simultaneous absorbance-transmission and fluorescence excitation-emission matrix method,” published in Methods and Applications in Fluorescence, Volume 6, Number 2 https://doi.org/10.1088/2050-6120/aaa818. Acquired with Aqualog.

To learn more about how A-TEEM molecular fingerprinting can help you with your application, email us at a-teem.us@horiba.com

A-TEEM for Wine

The wine industry was an early adopter of the Aqualog A-TEEM spectrometer to quantify wine phenolics, because this technique provides a simple and fast optical method that can save a large wine manufacturer a tremendous amount of time and money compared to more traditional analytical methods. Most of the work Aqualog is doing in the wine industry is protected under confidentiality agreements, however there is some information that can be shared about these applications.

Wine and Spirits Phenolic Classifications

Of the hundreds of different compounds that have been identified in grapes, it is the phenolic content of ripening grape berries that fundamentally determines the quality of a wine. The different classes of phenolics (anthocyanins, tannins, flavonols, catechins) affect the color, the mouthfeel, flavor and aroma to various extent, and most of these compounds fluoresce. The individual compounds comprising these classes of phenolics together give the wines their unique character. The A-TEEM data collected by Aqualog is used upstream and downstream to evaluate lot-to-lot, regional, and varietal characteristics in wine and grape juice.

Classical Least Squared (CLS) analysis of the wines and spirits

Classical Least Squared (CLS) analysis of the wines and spirits based upon a library of 9 phenolic compounds for illustrative purposes yields their relative contribution to the total phenolic fingerprint normalized to 100%. Significant differences between wines and spirits are reflected visibly in their phenolic compound fingerprints, acquired with Aqualog.

Subsequent application of a predetermined multivariate model, calibrated using a library of reference compounds, is the fastest and simplest technique to classify and compare wines and spirits to detect adulteration, spoilage, smoke taint, quantify SO2 treatment, and more.

To learn more about how A-TEEM molecular fingerprinting can help you with your application, email us at  a-teem.us@horiba.com

 

Fluorescence A-TEEMs for an Italian wine

Fluorescence A-TEEMs (A and B) and corresponding (C and D) Absorbance (OD) and % Transmittance spectra for an Italian wine sample before (A and C) and after (B and D) a one week oxidation treatment acquired on HORIBA Aqualog. (HORIBA App Note: FLSS-38, 2017)

A-TEEM for Cosmetics

EEM contour plot of in-vivo forearm skin

Figure 1. EEM contour plot of in-vivo forearm skin. Acquired with Aqualog.

In the cosmetics industry, the Aqualog A-TEEM spectrometer is used to investigate skin-state and sunscreen photoactivity. For skin characterization and sunscreen studies, a HORIBA Aqualog A-TEEM spectrometer is fitted with a remote fiber optic probe to acquire a fluorescence Excitation Emission Matrix (EEM) from the surface of the skin. Since this is a front face measurement from a solid surface, there is no absorbance measurement.

The endogenous fluorescence of skin is due to the presence of specific fluorophores (i.e. Porphyrins, Advanced Glycation End products (AGEs), Flavin Adenine Dinucleotide (FAD), Collagen, Elastin, Tryptophan, Tyrosine, NADH Pheomelanin, Eumelanin, components of Lipofuscin and Keratin, Hemoglobin (chromophore)). A Fluorescence EEM is shown to easily identify skin endogenous markers and characterize the interaction with cosmetic products.

Sunscreens protect the skin from the damaging effects of both UVA and UVB rays of natural light. They are characterized by the Sun Protection Factor (SPF). The HORIBA Aqualog can study photoactivity effects by acquiring an EEM before and after the application of a sunscreen. 

The main photo-process occurring in sunscreen is absorption: Sunscreens absorb into the outer skin layer (stratum corneum) and block the UV radiation from entering the inner skin layers by acting as optical filters, so the in-vivo fluorescence attenuation reflects the realized protective effects of the applied compounds. Although most of the high-energy UV photons are transformed, dispersed or absorbed by sunscreens, a certain amount of UV light will enter the epidermis.

Figure 2. Normalized EEM contour plot of in-vivo forearm skin after SPF20 a) and SPF50 b) sunscreen application, showing increasing filtering effects of transmission of excitation light relative to the unprotected skin EEM. Example acquired with Aqualog.

To learn more about how A-TEEM molecular fingerprinting can help you with your application, email us at a-teem.us@horiba.com.

A-TEEM for Olive Oil

In the food industry, the Aqualog A-TEEM spectrometer can be used to quantify olive oils with a simple, fast and sensitive optical method, compared to more traditional analytical methods.

Extra virgin olive oil (EVOO) is the highest quality rating for an olive oil, and consequently it is often adulterated with less expensive oils (Gurdeniz G.and  Ozen B.  Food Chemistry 116 (2009) 519–525).  EVOO’s are made of olives that are first cold pressed within hours after harvest,  without maceration or any extraction. All other fractions of olive oils are classified as not extra virgin olive oils. A-TEEM molecular fingerprinting can be used to easily identify and detect adulterated products.

Below are A-TEEM molecular fingerprints of seven commercial brands of EVOO-labelled olive oils and one brand of extra light (ELT).

A-TEEM fingerprints of commercial EVO labelled olive oils

Figure 1. Chlorophyll and degradation products can easily be seen in the A-TEEM fingerprints of some of these commercial EVOO-labelled olive oils, acquired with Aqualog.

Applying multivariate analysis to A-TEEM data provides more detailed analysis on the variations of these different brands.

3-D PARAFAC score plot of edible oils

Figure 2. 3-D PARAFAC score plot of edible oils. Note the grouping of EVOO oils in the bottom right corner: The upper left corner features vegetable oils and extra light olive oil. Curiously, the “G” brand EVOO and the “S” brand EVOO are distinct from the rest of the EVOO group, implying the presence of degradation products, as seem in their Aqualog A-TEEM fingerprints.

Investigating these samples further, by excluding the chlorophyll emission region, and applying a 2-D PARAFAC scoreplot, reveals additional information.

2-D PARAFAC scoreplot reveals that brand “A” moved to the region of brand “G” and brand “S”, while the rest of the EVO oils stayed together

Figure 3. 2-D PARAFAC scoreplot reveals that brand “A” moved to the region of brand “G” and brand “S”, while the rest of the EVOO oils stayed together in the bottom right corner of this scoreplot, as acquired with Aqualog.

The A-TEEM technique can also be challenged to quantitativelydetect limits of blending of an extra light olive oil into an extra virgin olive oil using a Partial Least Squares (PLS) analysis of A-TEEM fingerprints.

PLS analysis of mixtures of EVO oil

Figure 4. PLS analysis of mixtures of EVOO with increasing proportions of purified ELT oil reveals excellent R2 values for calibration and validation, acquired with Aqualog.

Even lot-to-lot variations can be detected within the same EVOO product of a single commercial brand.

3-D PARAFAC scoreplot showing lot to lot clustering from a prestigious EVOO brand

Figure 5. 3-D PARAFAC scoreplot showing lot-to-lot clustering from a prestigious brand purchased at different times and measured in a single session, acquired with Aqualog.

Focusing PARAFAC multivariate analysis of A-TEEM data to the chlorophyll fluorescence region (emission above 500 nm) also allows a quick analysis of the freshness of a given sample. PARAFAC readily resolves the A-TEEM fingerprint into two major components with distinct and characteristic fluorescence emission and excitation spectra (Fig. 6) for chlorophyll a and pheophytin.

Mode 3 loadings corresponding to fluorescence excitation spectra of Pheophytin and Chlorophyll a (Component 1 and Component 2 respectively)

Fig. 6. Mode 3 loadings corresponding to fluorescence excitation spectra of Pheophytin and Chlorophyll a (Component 1 and Component 2 respectively)

Fig. 7. illustrates a subsequent analysis of a set of EVOOs obtained from the current harvest obtained directly from the producers in Spain and Greece by Savantes (www.savantes.org) for the 5th Olive Oil Conference tasting program session held in NY in 2019, compared to store-bought samples from earlier harvests. The relative abundance of chlorophyll compared to its degradation product  pheophytin, can be related to the freshness of the product since Chlorophyll conversion to pheophytin starts to occur naturally over time immediately after packaging. It should be noted that the characteristic K232 and K270 values measured contemporaneously with EEM’s on the same sample by Aqualog remained within IOC guidelines (below 2.20 and 0.2 respectively) for all direct-sourced EVOOs but just for some of the store-bought ones.

Relative content of Chlorophyll and Pheophytin in direct sourced (2019 harvest) and store-bought EVOO’s (2018 and 2017 harvest).

Fig. 7. Relative content of chlorophyll and pheophytin in direct sourced (2019 harvest) and store-bought EVOOs (2018 and 2017 harvest). Avocado oil results are provided for comparison.

To learn more about how A-TEEM molecular fingerprinting can help you with your application, email us at a-teem.us@horiba.com

A-TEEM Products

Two HORIBA A-TEEM instruments to choose from

Aqualog-UV-800 featured with optional autosampler injector accessory

Aqualog Water Treatment Plant Analyzer

Aqualog-UV-800 featured with optional sipper accessory

Aqualog for Industrial QC/QA and Water Research

In 2010, HORIBA introduced the Aqualog® fluorescence and absorbance spectrometer, which offered millisecond CCD detection and real time fluorescence inner filter effect (IFE) correction. The Aqualog features ppb detection limit sensitivity, exquisite stray light rejection for the most demanding samples with highly scattering interference and full NIST-traceabilityfor complete intra- and inter-lab comparisons.

The Aqualog provides automated industrial accessories and solutions, such as a sipper for automatic sample extraction, and an autosampler for automatic vial and microwell plate sample extraction. Aqualog also offers an industrial HTML user dashboard that automatically presents the user with a variety of numbers for all analytes and predictors of interest, by automatically processing A-TEEM data sets through a multivariate analysis software model. The user pushes a button, and the interface presents them with analytical values.

The Aqualog is the gold standard for environmental water research and the system of choice for industrial QC/QA applications.

Duetta Fluorescence and Absorbance spectrometer

Duetta for Academic and Less Demanding Applications

Duetta™ is also a fluorescence and absorbance spectrometer based on the same A-TEEM technique as the Aqualog, However, unlike the Aqualog, which has been designed specifically for industrial applications with the right level of automation, the Duetta was designed for academic, and more general applications.

Request for Information

Do you have any questions or requests? Use this form to contact our specialists. * These fields are mandatory.

Browse Products

Fast-01
More Fast-01

Aqualog A-TEEM Autosampler Accessory

Aqualog
More Aqualog

Water Treatment Plant Analyzer

Duetta
More Duetta

Fluorescence and Absorbance Spectrometer