ISO/TR 19693:2018

Surface chemical analysis — Characterization of functional glass substrates for biosensing applications

ISO/TR 19693:2018 gives examples of how methods of surface chemical analysis in the scope of ISO TC 201 are useful to characterize the nature of substrates used to produce biosensing devices. Successful characterization will give the opportunity for a better understanding of aspects of surface chemistries and reactions at each step of production influencing the overall performance of the final device, for example a microarray. The steps of preparation are the activation of the substrate by immobilization of linker molecules and the functionalization of the activated substrate with biomolecules required for specific biosensing, the so-called probes. Herein, a focus is set on silane-based functionalization of glass slides, a critical production step for subsequent immobilization of probe molecules. Those probes are used for sensing of biological recognition events. The silanization process has been selected because it is one of the most popular in biosensor production today. ISO/TR 19693:2018 gives an overview of methods, strategies and guidance to identify possible sources of problems related to substrates, device production steps (cleaning, activation and chemical modification) and shelf-life (storage conditions and ageing). It is particularly relevant for surface chemical analysts characterizing glass-based biosensors, as well as developers or quality managers in the biosensing device production community. Based on quantitative and qualitative surface chemical analysis, strategies for identifying the cause of poor performance during device manufacturing can be developed and implemented. This document shows how far the light may shine today and possible starting points for more specific activities of ISO/TC 201 in the future, which end in standardized procedures for measurements. No specific protocols on processing are discussed in this document. To learn more about protocols the reader is referred to specialized literature, see for example References [1] to [9].

Analyse chimique des surfaces — Caractérisation de substrats de verre fonctionnels pour les applications de biodétection

General Information

• Status: Published
• Publication Date: 08-Feb-2018
• ICS: 71.040.40 - Chemical analysis
• Technical Committee: ISO/TC 201 - Surface chemical analysis
• Drafting Committee: ISO/TC 201/WG 4 - Surface characterization of biological materials
• Current Stage: 6060 - International Standard published
• Completion Date: 09-Feb-2018

Standards Content (Sample)

TECHNICAL ISO/TR
REPORT 19693
First edition
2018-02
Surface chemical analysis —
Characterization of functional glass
substrates for biosensing applications
Analyse chimique des surfaces — Caractérisation de substrats de
verre fonctionnels pour les applications de biodétection
Reference number
ISO/TR 19693:2018(E)
©
ISO 2018

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ISO/TR 19693:2018(E)

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ISO/TR 19693:2018(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 2
5 Characterization of substrates for biosensors by surface chemical analysis .2
5.1 Introduction . 2
5.2 Surface chemical analysis (SCA) . 3
5.2.1 General. 3
5.2.2 X-ray photoelectron spectroscopy (XPS) . 3
5.2.3 Time-of-flight secondary ion mass spectrometry (ToF-SIMS) . 4
5.2.4 Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy . 4
5.2.5 Other methods . 5
5.3 Characterization of microarray glass substrates . 6
5.3.1 Glass surface composition . 6
5.3.2 Characterization of cleaned and pre-activated glass slides . 7
5.4 Characterization of functional silane films formation on glass substrates . 8
5.5 Stability and shelf-life of aminosilane-based biosensing platforms on glass .12
5.6 Other types of functionalized glass slides .17
6 Concluding remarks .21
Annex A (informative) Study of pre-cleaned commercial glass slides .25
Bibliography .28
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ISO/TR 19693:2018(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
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URL: www .iso .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 201, Surface chemical analysis.
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Introduction
Sensing devices based on immobilized biomolecules on solid substrates are a steadily growing market
in personalized medicine and point of care (POC) diagnostics, which are becoming tremendously
important for our society. Precise knowledge of these biointerfaces is a prerequisite for a reliable and
proper functionality of such biosensing devices. This kind of knowledge includes surface composition,
surface chemistry (e.g. functional groups, surface species), surface structure/morphology, in-depth
compositional profiles and film thickness, which can be obtained by a thorough physico-chemical
characterization using surface chemical analysis.
This report on surface chemical analysis of glass substrates for biosensors prepared by ISO/TC 201/WG 4
has been prepared in coordination with the overall characterization needs identified by experts in TC 201.
This document describes the information that can be obtained by the different analytical techniques
and examines how this information can be used to understand and solve important questions and
challenges in the biosensor production process.
With that focus, consideration of
— bulk composition;
— surface composition;
— cleanliness;
— wettability;
— reactivity; and
— stability
are relevant and (in an ideal case) should be known for each component, i.e. substrate, functional
coating/layer and biomolecular probes, of a reliable biosensing device.
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TECHNICAL REPORT ISO/TR 19693:2018(E)
Surface chemical analysis — Characterization of functional
glass substrates for biosensing applications
1 Scope
This document gives examples of how methods of surface chemical analysis in the scope of ISO TC 201
are useful to characterize the nature of substrates used to produce biosensing devices. Successful
characterization will give the opportunity for a better understanding of aspects of surface chemistries
and reactions at each step of production influencing the overall performance of the final device, for
example a microarray. The steps of preparation are the activation of the substrate by immobilization
of linker molecules and the functionalization of the activated substrate with biomolecules required for
specific biosensing, the so-called probes.
Herein, a focus is set on silane-based functionalization of glass slides, a critical production step
for subsequent immobilization of probe molecules. Those probes are used for sensing of biological
recognition events. The silanization process has been selected because it is one of the most popular in
biosensor production today.
This document gives an overview of methods, strategies and guidance to identify possible sources of
problems related to substrates, device production steps (cleaning, activation and chemical modification)
and shelf-life (storage conditions and ageing). It is particularly relevant for surface chemical analysts
characterizing glass-based biosensors, as well as developers or quality managers in the biosensing
device production community. Based on quantitative and qualitative surface chemical analysis,
strategies for identifying the cause of poor performance during device manufacturing can be developed
and implemented. This document shows how far the light may shine today and possible starting points
for more specific activities of ISO/TC 201 in the future, which end in standardized procedures for
measurements.
No specific protocols on processing are discussed in this document. To learn more about protocols the
reader is referred to specialized literature, see for example References [1] to [9].
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 18115-1:2013Surface chemical analysis — Vocabulary — Part 1: General terms and terms used in
spectroscopy
ISO 18115-2:2013Surface chemical analysis — Vocabulary — Part 2: Terms used in scanning-probe
microscopy
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 18115-1:2013 and
ISO 18115-2:2013 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
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4 Symbols and abbreviated terms
AFM atomic force microscopy
APDMES 3-aminopropyldimethylethoxysilane
APS 3-aminopropylsilane
APTES 3-aminopropyltriethoxysilane
APTMS 3-aminopropyltrimethoxysilane
FT-IR Fourier transform infrared spectroscopy
LEIS low energy ion scattering
MEIS medium energy ion scattering
NEXAFS near-edge X-ray absorption fine structure
PCA principle-component analysis
SCA surface chemical analysis
SFG sum frequency generation spectroscopy
STM scanning tunnelling microscope
ToF-SIMS time-of-flight secondary ion mass spectrometry
TXRF total reflection X-ray fluorescence
WCA water contact angle
XPS X-ray photoelectron spectroscopy
5 Characterization of substrates for biosensors by surface chemical analysis
5.1 Introduction
Biosensing as a concept utilizes a biological or biomolecular recognition event that is transduced into
a measurable signal (electrical, optical, chemical or thermal). Therefore in many biosensor devices, a
biomolecular system acting as probe is immobilized to a solid support to capture the specific target
[7][10–12]
(analyte) from the sample giving a biochemical reaction that is transformed into a signal .
A special type of biosensing application is the microarray, a high-throughput analytical tool for studying
biological processes enabling quantitative and simultaneous analyses of a large number of biomolecular
interactions in a very short time. A typical microarray consists of biomolecules arranged in single spots
in a miniaturized, spatially defined fashion and attached to a solid surface (see Figure 1).
Figure 1 — Example of a microarray with subarrays
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Microarrays are essential tools for high-throughput screening and detection in biochemical,
bioanalytical, medical or diagnostic research and especially in the continuously growing field of omics
[13–15]
(genomics, proteomics, glycomics and metallomics) . However, there is still a need for approaches
to sidestep current problems associated with the repeatability, reproducibility and quantification of DNA
and peptide arrays by understanding the factors that contribute to unreliable performance. Analytical
tools are required which may help to understand the origin of variability in array performance and
[16]
mitigate such variability . Substantial improvement in reliability and reproducibility of biosensors
will support clinical acceptance of biosensor-based diagnostic devices.
Glass is one of the most frequently used substrates for biosensor and microarray fabrication because of
its low price, availability, stability (against temperature, many chemicals and biological materials), low
fluorescence, flatness, non-porosity and manifold possibilities of surface modifications.
Most glass substrates used in biosensor manufacturing are coated first with a functional silane layer.
Then, these silane films are used directly or after activation for binding the probe molecules of interest.
Careful quality control for the cleaning, pre-activation and silanization steps is mandatory, especially
[17–19]
for identifying contaminants and proper characterization of the applied coatings .
In this report, well-known approaches of surface chemical analysis that can be used to control
every single step of glass-based biosensor production are introduced to developers of biochips and
quality managers in their production. Equally, surface chemical analysts who have a specific need to
characterize glass-based biosensors are addressed by this report.
[20]
Routinely applied simple methods for surface analysis, such as water contact angle measurements ,
have an intrinsic limitation of giving only macroscopic summary information of the analysed surfaces.
In contrast to that, a multi-method approach of surface analytical techniques can yield detailed
information about surface chemistry, species and composition, and subsequently, a combination of
vacuum-based surface characterization techniques bears great potential for identifying issues in
manufacture and quality control.
5.2 Surface chemical analysis (SCA)
5.2.1 General
This subclause gives a short overview of surface characterization techniques currently covered
by TC 201 which are fit for purpose. It is prepared to inform users from the biosensing community
unfamiliar with those methods. For details on individual techniques the reader is referred to specific
[21][29]
textbooks and other literature .
5.2.2 X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS) is a powerful tool for surface chemical analysis, as it gives
specific elemental as well as chemical information with only a few restrictions concerning the specimen.
In XPS the specimen is irradiated with X-rays under ultra-high vacuum (UHV) conditions. Due to
the photoelectric effect the surface is emitting photoelectrons. The kinetic energy of the detected
photoelectrons is characteristic of the individual constituent element acting as emitting atom. A
quantitative elemental analysis of the probed surface layer is possible, except for H and He. Details of
specific binding states of the emitting atoms are also possible because the binding energy of the emitted
photoelectron is correlated with its binding situation.
A characteristic chemical shift is observed in many cases. By analysis of highly resolved spectra,
quantitative information on binding states of the elements in the probed surface can be discovered.
Coexisting chemical species in the sample can be often differentiated from each other because today
most laboratory XP spectrometers are using monochromatized X-rays, which enable high energy
resolution. Imaging photoelectron spectroscopy can also be carried out. In this case contrast results
either from different elemental distributions or different chemical shifts for spectral components of
a highly resolved core-level spectrum of one element. An ultimate lateral resolution of ~10 µm can be
[30]
reached using recent instruments. Typical sensitivities in XPS are in the range of 0,1 at% to 1,0 at% .
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With a typical Al Kα X-ray source the information depth is in the order of 10 nm and depends on the
[22][27]
photoemission signal used .
Because modern XPS instruments are equipped with charge neutralizers, bare and functionalized glass
slides can be analysed without any conductive coating of the samples.
5.2.3 Time-of-flight secondary ion mass spectrometry (ToF-SIMS)
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is secondary ion mass spectrometry
(SIMS) using a time-of-flight (ToF) mass analyser. SIMS is based on primary ion bombardment of the
sample in UHV with primary ion energies of some keV. During that process the energy of the primary
ions is transferred by atomic collisions into the solid, causing a cascade of collisions. Since some of the
energy is transferred back to the surface, atomic and molecular fragments – but also complete molecules
(masses up to 10 000 u) – are ejected from the surface topmost layers, which can be detected if they
were ionized. Fragment patterns in SIMS spectra can often be correlated with specific compounds
present in the probed surface area. Careful analysis of the detected masses of desorbed secondary ions
in SIMS spectra reveals details on elemental and molecular composition of the analysed surfaces.
For chemical analysis SIMS is operated in the “static mode”, in which the material surface is sputtered
at a sufficiently low rate that the original surface is insignificantly damaged during the analysis.
Distribution images with lateral resolution in the sub-µm region are obtained if focused primary ion
beams are used. SIMS is more surface sensitive than XPS because the detected secondary ions are
emitted from the topmost surface (1-3 monolayers) and ppm sensitivities can be reached for selected
elements.
ToF mass spectrometers are characterized by high transmission and sensitivity with a broad application
range. Different operation modes are possible surface spectroscopy, imaging and depth profiling.
Because modern ToF-SIMS instruments are equipped with charge neutralizers, bare and functionalized
glass slides can be analysed without any conductive coating of the samples.
ToF-SIMS experiments usually result in huge data sets. Fragmentation often leads to hundreds of peaks
in a spectrum. In most cases the relevance of a single peak is unknown, but sometimes characteristic
fragment patterns are known and (quasi)molecular peaks are identified. Therefore, data reduction
methods, for example principal-component analysis (PCA), are used to extract all relevant information.
Scores and loadings plots delivered by PCA help to differentiate chemical states, to identify characteristic
[23–26]
peaks and to retrieve semi-quantitative information .
5.2.4 Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy
Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy provides chemical state information
that is complementary to XPS, concerning the chemical binding and quantity of molecules on a surface.
In the electron yield mode NEXAFS spectroscopy has the same information depth as XPS. Saturated and
unsaturated bonds in organic matter can be easily differentiated by NEXAFS spectroscopy.
Synchrotron light is polarized and utilizing angle-resolved NEXAFS spectroscopy is a tool to determine
preferential orientations of molecules or specific functional groups on the surface in a similar way
to what can be done in IR spectroscopy. In a NEXAFS experiment the energy of synchrotron light is
stepwise varied across the absorption edge, for example for a K-edge of carbon or nitrogen. By doing
so resonant transitions from a core level, for example C 1s or N 1s, into molecule-specific unoccupied
molecular states, for example the LUMO, can be observed in the X-ray absorption spectra as σ and π
resonances. These spectral features make NEXAFS spectroscopy a very useful method to characterize
a sample on a molecular level, but due to the necessity of a synchrotron source it is not a routine surface
[28][29]
analytical technique like XPS or ToF-SIMS .
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5.2.5 Other methods
5.2.5.1 General
In this document the main focus is on XPS, SIMS and NEXAFS, but there are complementary techniques
that are often used in characterization of functional glass substrates for biosensor applications. These
include spectroscopic ellipsometry, Fourier transform infrared spectroscopy (FT-IR), Raman, sum
frequency generation spectroscopy (SFG), fluorescence spectroscopy, atomic force microscopy (AFM),
scanning tunnelling microscope (STM), low energy ion scattering (LEIS), medium energy ion scattering
(MEIS), total reflection X-ray fluorescence (TXRF) and water contact angle (WCA), to name only the
most relevant ones. Some of them are less known to the community addressed by this document and
are explained in some detail in this subclause.
[25][26][31][32]
5.2.5.2 Ion scattering (LEIS and MEIS)
LEIS delivers quantitative information on the atomic composition of the outermost atomic layer of a solid
sample. In LEIS, a beam of low energy noble gas ions with a kinetic energy of a few keV is used to probe
the surface. The energy of each backscattered ion is characteristic for the mass of the corresponding
collision partner, which is here a surface atom. The energy of the backscattered ions is measured by
an energy analyser and element analysis of the surface is enabled. Ions that are scattered from depths
up to 10 nm can also be detected and lead to a signal that is characteristic for the particular in-depth
composition of the respective elemental, and layer thickness can also be determined. Because the LEIS
scattering regime includes only negligible sputtering, the chemistry of biosensing platforms can be
studied and depth profiling is non-destructive.
MEIS basically uses the same approach as LEIS but uses ions of higher energies around 100 keV.
MEIS is a technique that allows probing film composition with sub-nanometre depth resolution and
is preferentially applied to oxide layers used in microelectronics. It can be easily used to study bare
substrates as glass slides used for bio sensing devices. So far there is only one MEIS application in the
[32]
literature where an organometallic film is studied .
5.2.5.3 Spectroscopic ellipsometry
Ellipsometry is capable of measuring changes in film thickness with sub-nanometre precision, if there
is sufficient knowledge of the refractive indices of the materials in the substrate and overlayer. For
organic films on glass, the refractive indices of the two materials are very similar in the visible region,
and therefore measurements of overlayer thickness are very challenging. Ellipsometry in regions of the
infrared, where the organic film has significantly different optical properties to glass, shows excellent
[26][28][33][35]
potential .
5.2.5.4 Total reflection X-ray fluorescence
TXRF utilizes extremely low-angle X-ray excitation of flat surfaces of bare or functionalized glass slides
to obtain the concentration of surface contaminants and elements in functional layers. Using total
reflection conditions (incident angle of the X-ray beam typically as small as 0,05°), excitation is limited
to the outermost surface of the sample. By using monochromatic synchrotron radiation in the soft
X-ray range and an ultra-high vacuum setup including windowless detectors, even light elements on a
substrate surface, for example carbon or nitrogen, can be excited effectively. That high experimental
[36–41]
effort facilitates quantitative TXRF analyses of organic nanolayers on flat substrates .
Recently traceable TXRF has been shown to provide direct access to the mass-per-unit area of selected
elements in a thin surface layer. The mass-per-unit area of nitrogen was determined for aminosilane
functionalized Si wafer substrates, which can be used to determine the number of amino groups on the
surface. Based on this, XPS was calibrated and absolute and traceable quantification of surface-bound
[42]
organic molecules on silicon oxide surfaces by XPS enabled .
[43–45] [25][26]
Methods such as fluorescence spectroscopy , vibration spectroscopy (FT-IR and Raman)
[28][46] [25][26][28] [28]
, scanning probe microscopy (e.g. AFM, STM) and water contact angle goniometry
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are widely used in the community addressed by this document and references to relevant reviews and
textbooks are referenced in this subclause.
5.3 Characterization of microarray glass substrates
5.3.1 Glass surface composition
An important but often disregarded or ignored factor influencing the whole biosensing experiment is
the chemical composition of the glass substrate. For example, glass with high contents of sodium and
phosphates is very challenging. Non-bonding ions such as phosphates and alkali metal ions are known
to catalyse Si-O bond breakage and redistribution. Additionally, contaminants from the bulk tend to
segregate to the surface. Therefore, these unwanted species have to be removed before any further
[47]
processing. Species can be identified and their successful removal verified by surface analysis .
The elemental composition in regions close to the surface (within ca. 10 nm) of glass samples can
be measured by XPS according to, for example, ISO 18118:2015. From that information it is possible
to differentiate between different glass substrates, such as soda-lime, phosphate or borosilicate
[48][49]
glasses .
At BAM Federal Institute for Materials Research and Testing, the surface composition of glass slides from
different vendors used for microarray applications were compared using XPS. Silicon and oxygen were
the major constituents in all the glass slides. Slides made of soda-lime silicate glass are characterized by
significant amounts of Na, Mg and Ca. Borosilicate slides exhibit boron as one of the main constituents
combined with only low amounts of sodium. In addition, minor amounts of aluminium, nitrogen and
potassium were found on all slides. Samples made by the float glass process show characteristic tin
peaks on one side. (For more details see Annex A, Table A.1.)
Alterations to the glass formulation of commercial soda-lime glass substrates may have dramatic
[49]
effects on biosensor performance, as described by North et al. , who observed a significant change in
antibody immobilization with glass composition. After a systematic screening of the bare glass slides
using water contact angle measurements (WCA), fluorescence spectroscopy and XPS, the performance
of microarrays expressed as antibody immobilization efficiencies was related to individual chemical
compositions, specifically a variation of the magnesium content, and surface morphologies expressed
as roughness of the used glass slides (see Figure 2). XPS was used to identify elemental disparities in
the glass surface of different commercial soda-lime slides.
NOTE Slide A (red; high magnesium) and slide B (blue; low magnesium) by contact angle measurements (top
level), XPS (centre), atomic force microscopy (middle level) and functional binding assay (lower level) reveal a
[7][49]
significant correlation between magnesium content, surface roughness and bioimmobilization efficacy .
6 © ISO 2018 – All rights
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