Encyclopedia of Microfluidics and Nanofluidics

Living Edition
| Editors: Dongqing Li

Infrared Imaging and Mapping for Biosensors

  • Karsten HinrichsEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-27758-0_690-5



Infrared (IR) imaging or mapping techniques are meant here as those that potentially can be used for biosensors and their characterization. Far-field and near-field infrared spectroscopy involve concepts of measurement in multifold transmission or reflection modes [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. The mid-infrared (MIR) spectral range is defined from 2.5 to 16 μm (IR in [1]). For the production of laterally resolved IR images [2, 3], typically a two-dimensional sample movement stage is employed for defined lateral movements of the sample, whereby for every position a measurement with a single detector is performed or alternatively the image is obtained in single shot involving a detector array (e.g., focal plane array in [2]). A principle optical scheme for microscopy is shown in Fig. 1. Typical spectrometers used for the MIR range are Fourier transform infrared (FTIR) spectrometers; however, for specific purposes also infrared grating spectrometers are taken. The standard spectral MIR source in the lab is the glowbar, whereas synchrotron or laser radiation can be used as brilliant radiation source. Optical parametric oscillator (OPO) sources, difference-frequency generation (DFG) sources, gas, or quantum cascade lasers (QCL) are often used in the laboratory [16], some of which are tunable or available as broadband sources in certain spectral ranges. Recently the application of such lasers provides new possibilities in IR mapping techniques in classical far-field spectroscopy but also in near-field spectroscopy.
Fig. 1

Principle of infrared mapping experiment for biosensors in a microscopic setup in reflection and transmission mode. When the single detector is replaced by a detector array, imaging becomes available. For spectroscopic experiments, typically a FTIR spectrometer is introduced after the broadband source (e.g., glowbar). Alternatively tunable or single wavelength lasers can be directly attached. In other IR spectroscopic techniques, the Cassegrain objective might be replaced by other focusing elements like lenses or parabolic mirrors at multiple angles of incidence. In specific measurement geometries also optical fibers or total reflecting prisms are used


Many optical techniques could potentially be used for biosensors and their characterization [1]. Among the label-free optical vibrational spectroscopies are Raman spectroscopy (RS) and infrared spectroscopy [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13], which are frequently used as fingerprinting methods for characterization of thin films of biological compounds and materials as well as biosensors. In comparison to the electronic bands in the visible, for the IR spectral range, absorption bands of molecular vibrations can often give a higher spectral contrast. For example, for self-assembled DNA probes on a gold surface, the distribution of OH-bands was imaged by IR microscopy over a 5 × 5 mm region via the corresponding absorption band centered at 3,181 cm−1 [13]. The interpretation of absorption bands can deliver, for example, thickness, molecular identification, molecular orientation, molecular interaction, and composition. For the IR spectral range, standard far-field spectroscopy methods exist in transmission or reflection modes as shown in Fig. 2. Concepts of measurements involving multiple incidence angles and the use of polarizers provide further information, for example, in external reflection spectroscopy (e.g., reflection absorption IR spectroscopy (RAIRS) [4] and IR spectroscopic ellipsometry (IRSE) [15]). Ellipsometry measures the elliptical polarization of light and has the advantage that it provides two or more independent measurement parameters. For certain applications, the sensitivity can be increased by IR internal reflection spectroscopy (e.g., attenuated total reflection (ATR) [1, 6]) and implementation of surface-enhanced infrared absorption (SEIRA) substrates. For higher lateral resolutions, all the methods can be in principle combined with an IR microscopic [2] setup. All of the methods are typically noninvasive and could potentially be used for biosensor studies. Additionally they offer the possibility to be coupled with imaging techniques. Even though there is an emerging potential for IR spectroscopy for studying biosensors [5, 7, 14] and IR imaging of biomedical samples [8, 9, 10, 13, 17], only a few studies with respect to the IR imaging of biosensors have been published up to now [5]. Figure 3a shows the principle of IR synchrotron mapping ellipsometry and Fig. 3b an experimental example of biosensor characterization by IR synchrotron mapping ellipsometry. Each stage of a surface modification in the process of biofunctionalization and biosensing can be proved by IRSE spectra. As an example, Fig. 4a displays a schematic for antibody immobilization onto maleimidobenzene modified substrates for which the specific functionalization with imide rings allows efficient binding of the cys-peptide to the surface. Characteristic IR spectra taken during the course of functionalization of a silicon surface with maleimidobenzene, the modification with cys-peptide, and coupling of an antibody to the surface are shown in Fig. 4b [14].
Fig. 2

Schematic of transmission, external reflection, and internal reflection modes at multiple incidence angles φ. For the internal reflection, a single reflection in attenuated total reflection (ATR) geometry is shown exemplarily. The polarization of the E-field of the incident radiation parallel (p-polarized) and perpendicular to the incidence plane (s-polarized) is marked in the transmission and external reflection geometry. Typical measurement parameters are the absorbance (reflectance absorbance) which can be calculated for s- and p-polarized radiation by −log (I1/I0), where I1 represents the transmitted (reflected) intensity from an adsorbate-covered substrate, while I0 is the transmitted (reflected) intensity from the bare substrate

Fig. 3

(a) Principle of IR synchrotron mapping ellipsometry. Due to reflection at the sample, the polarization of the radiation is changed. For example, the incident linearly polarized radiation becomes elliptically polarized. The sample is moved by a two-dimensional mapping table, and a spectrum is taken for every probed spot. The ellipsometric parameters defined by the quantity ρ, which is the ratio of the complex reflection coefficients r p and r s , are measured for every spot. Δ is the phase shift and tan Ψ the amplitude ratio of p- and s-polarized reflection coefficients. (p-polarized is defined as parallel and s-polarized as perpendicular to the incidence plane.) (b) Three-dimensional presentation and contour plots of 7 × 7 mm map of ellipsometric (tan Ψ) band amplitudes of a peptide band at 1547 cm−1 (Adapted from [5]). The peptide Cys-GCN4 was immobilized on a linker-covered silicon wafer. In principle electronic recognition of the specific adsorption site could serve for fast analysis of the samples under investigation [5]

Fig. 4

(a) Schematic for antibody immobilization onto maleimidobenzene functionalized substrates. The specific functionalization with imide rings allows efficient binding of the cys-peptide at the surface. (b) Referenced infrared (tan Ψ) spectra of maleimidobenzene modified Si surfaces, their modification with cys-peptide and coupling with specific antibody (Adapted from [14]). A detailed discussion can be found in [14]

Basic Methodology

Laterally resolved images of biosensors can be produced by various IR technologies. Besides the optimization of measurement time, important key properties of the imaging techniques in the MIR range are:
  • Lateral resolution. When brilliant light sources like lasers or synchrotron radiation are used, the lateral resolution typically is diffraction limited in the range of a few micrometers for IR microscopy [2, 11]. This can be improved by orders of magnitude with near-field IR microscopy [12], which enables resolution down to a few tens of nanometers, and spectroscopic studies by tuning of lasers [18] or use of a broadband synchrotron or laser source. IRSE biochip characterization with lateral resolution down to approximately 200 × 400 μm is possible.

  • Sensitivity. For SNIM, RAIRS, ATR, and IRSE, the high sensitivity allows detection of nanometer thick biomolecular films.

  • Quantification of experimental results. Reliable quantification of optical spectra and signals can be performed by optical modeling of RAIRS, ATR, and IRSE spectra. Here, IRSE has some advantages because it delivers up to three method-independent parameters (phase shift and amplitude ratio of s- and p-polarized reflection coefficients and the polarization degree). For SNIM quantitative evaluation is more complex than for the other methods and is dependent on the geometry of the SNIM experiment.

Key Research Findings

Sensors in Biochemistry and Biomedical Applications

Infrared imaging and mapping can be used for design of new functional sensing templates based on smart thin films and surfaces. Label-free observation of vibrational bands allows specific identification of molecular bonds, molecular groups, and materials. The interpretation of IR signals enables the analysis of the binding chemistry, structure, and molecular interactions and can give quantitative conclusions on the amount of adsorbed material. In situ characterization might be used for monitoring of chemical changes as well as molecule adsorption in dependence of growth conditions and external stimuli.

Sensors in Microfluidic Devices

In combination with reversible and smart templates, analysis of liquids in a flow cell can be performed.

Sensors in Combination with SEIRA

Specific substrates like, e.g., metallic island films or metallic wires, may show a SEIRA effect. In combination with a device, this effect can enhance the detection sensitivity.

Future Directions

With the development of infrared lasers (e.g., quantum cascade lasers) having spectral tunability or are available as broadband source in the MIR spectral range, the implementation of such lasers in infrared measurement technology is increasing. Due to possible higher lateral resolution and faster measurement protocols, such techniques are expected to be especially valuable for new sensing systems which include functional surfaces and thin films sensitive on external stimuli (pH, temperature, electric field, light, etc.). Such smart interfaces can be combined with techniques either sensing or releasing specific analytes. Using in situ IR spectroscopy studies in liquid environment is feasible, providing a high potential for bioanalytical and biomedical applications.



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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Leibniz-Institut für Analytische Wissenschaften - ISAS - e.VBerlinGermany