For Peer Review
1
Integrated optical devices for lab-on-a-chip biosensing
applications
M.-Carmen Estevez, Mar Alvarez and Laura M. Lechuga*
Nanobiosensors and Bioanalytical Applications Group, Research Center on
Nanoscience and Nanotechnology (CSIC) & CIBER-BBN. Campus UAB, 08193
Bellaterra, Barcelona, Spain
*Email:
Laura.lechuga@cin2.es
Phone: +34-935868012
Fax: +34-935868020
Abstract. The application of portable, easy-to-use and highly sensitive lab-on-a-
chip biosensing devices for real-time diagnosis could offer significant advantages
over current analytical methods. Integrated optics-based biosensors have become
the most suitable technology for lab-on-chip integration due to their ability for
miniaturization, their extreme sensitivity, robustness, reliability, and their potential
for multiplexing and mass production at low cost. This review provides an extended
overview of the state-of-the-art in integrated photonic biosensors technology
including interferometers, grating couplers, microring resonators, photonic crystals
and other novel nanophotonic transducers. Particular emphasis has been placed on
describing their real biosensing applications and wherever possible a comparison of
the sensing performances between each type of device is included. The way towards
achieving operative lab-on-a-chip platform incorporating the photonic biosensors is
also reviewed. Concluding remarks regarding the future prospects and potential
impact of this technology are also provided.
Short title: M.C. Estevez, M. Alvarez and L.M. Lechuga: Integrated optical
biosensors.
PACS: 87.85.fk, 42.82.Et, 07.07.Df, 07.07.Mp, 07.60.Ly, 87.85.Rs
Keywords: Photonic biosensors, evanescent wave detection, lab-on-a-chip,
biofunctionalization, silicon photonics.
Page 1 of 56
Wiley - VCH
Laser & Photonics Reviews
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
2
1. Introduction
Biosensors are devices able to detect a specific substance by converting the recognition from a
biological entity (i.e. DNA, antibody, enzyme,...) into an electrical signal that can be further
processed and related to the concentration of the substance under analysis. Biosensors can
provide selective, sensitive, fast, direct and cost-effective analyses. In addition, they can
perform tests in real-time without using fluorescent labels or amplification steps and with a
minimum volume of samples and reagents [1]. As compared to standard techniques which are
usually time-consuming, expensive and require labelling and trained personal, biosensing
technology offers clear advantages.
Lab-on-a-chip (LOC) are miniaturized devices in which all functionalities are integrated in the
same platform, from sample preparation to signal delivery [2]. Ideally a LOC device should
contain enough hard wired intelligence and robustness to be used by non-skilled personal and
should deliver the results directly to a central monitoring station. It is clear that achieving a
small, portable and easy-to-use lab-on-a-chip device for diagnostics could offer significant
advantages over standard methods. Although significant progress has been accomplished at the
LOC field during last years, very few stand-alone devices have emerged [3]. Most of current
devices are simple planar microfluidic devices which do not incorporate the detection and after
the reaction has taken place, the read-out must be done with complex instrumentation in
laboratory settings. That is the main reason why incorporating “on-chip” detection by using
biosensors is a new technology that shows great promise. Main application fields of this
technology can be clinical diagnostics, environmental monitoring, chemical and biological
warfare surveillance, food industry and veterinary and industrial process control, among others.
By using this advanced technology, diagnosis in developing countries could become an
important achievement for the near future.
Photonic biosensors are well-established technologies for the sensitive monitoring of molecular
interactions. They could afford the requirements for the “on-chip” detection in lab-on-a-chip
platforms due to their outstanding characteristics of sensitivity, label-free and real-time
detection. The detection principle of most optical biosensors is based on the evanescent field
detection. In the evanescent wave mechanism (see Figure 1), a bioreceptor layer is immobilized
onto the surface of a waveguide; the exposure to the partner analyte produces a biomolecular
interaction affecting the guiding properties of the waveguide (concretely, a variation of the
refractive index) via the modification through the evanescent field. The variation of the
refractive index can be evaluated by any of the waveguiding optical properties (intensity, phase,
Page 2 of 56
Wiley - VCH
Laser & Photonics Reviews
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
3
resonant momentum, polarization,...) and this variation can be correlated with the concentration
of the analyte and with the affinity constant of the interaction, resulting in a quantitative value
of the interaction.
As the evanescent wave decays exponentially while it penetrates into the outer medium, it only
detects changes taking place on the surface of the waveguide, since the intensity of the
evanescent field is much higher in this particular region. For most waveguide systems, this
decay length is on the order of 0.1-1 µm. For that reason, it is not necessary to carry out a prior
separation of non-specific components (as in conventional analyses) because any change in the
bulk solution will hardly affect the sensor response. Therefore, the most significant advantages
of evanescent-based mechanism are the highly sensitive and specific label-free detection of
target molecules or biochemical reactions in real time, with reduced nonspecific binding, which
makes this detection mechanism one of the most useful for detection of targets in complex real
samples.
The most common optical evanescent wave biosensor is the Surface Plasmon Resonance (SPR)
device [4] based on the variation of the reflectivity on a metallic layer in close contact with a
dielectric media. The SPR biosensor has been widely developed and commercialized and
hundreds of publications have demonstrated its outstanding performance to evaluate complex
biosensing interactions [4]. But SPR sensor has a relatively large size and its miniaturization in
lab-on-chip platforms is complex. Moreover, the sensitivity is usually limited to the nanomolar
range, which is extremely useful in diverse applications [5-7]
but not enough for applications
requiring lower detection levels (pM-fM or even single-molecule), which are usual in the
clinical practice.
Photonic sensors based on integrated optics (IO) could solve the aforementioned SPR problems,
as they can be easily miniaturized and they offer high potential for chip integration; moreover,
by using evanescent wave as detection mechanism, sensitivities can be extremely high (pM in a
label-free scheme). Moreover, integrated optics allows a great flexibility in the materials and
structures selection and fabrication of arrays of sensors with the same characteristics within the
same chip for multiplexing analysis can be afforded. Materials employed in IO devices are Si,
Si
3
N
4
, SiON, SiO
2
or polymers, and techniques such as ion-diffusion in glass, chemical vapour
deposition, spin-coating, nanoimprinting, electron beam lithography, etc are commonly
employed for the fabrication. By using silicon photonics technology, additional advantages such
as robustness, reliability, low power consumption and potential for mass production with
consequent reduction of production costs are added. Other technologies as III-V or lithium
Page 3 of 56
Wiley - VCH
Laser & Photonics Reviews
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
4
niobate, often used in optical telecommunications, have shown less suitability for IO biosensors
as they are more complex and expensive.
Most of the refractive index IO sensors rely on the induced changes of the effective refractive
index by the biomolecular interaction in the evanescent area. In order to achieve highest
sensitivity, the waveguiding structure must be optimized looking for a maximum change of the
effective refractive index due to the sensing biolayer. Sensitivity is a complex parameter
involving geometry, material and working wavelength of the waveguide, and also other aspects
such as the chemical activation of the surface, the biofunctionalization method and the
resolution and noise of the optical read-out system. In addition, fluidics must be taken into
account (i.e. flow cell volume, way of sample injection, diffusion, dispersion). Each of these
aspects must be properly designed and optimized before delivering a functional biosensor
device. Integrated optical sensors such as grating-couplers, interferometers, photonic crystal,
microring resonators, slot waveguides or silicon wires have been extensively studied in the last
years [8-12],
but so far only few of them are commercially available.
One of the main advantages of the IO technology is the possibility to integrate all the functions
(chemical, optical, microfludics and electronics) in one single platform offering an ideal
solution for the implementation of truly lab-on-a-chip devices. This area is still in its infancy,
but remarkable progress has been achieved during last years [3,13]. This is reflected in the
increasing amount of publications addressing new or optimized sensing configurations.
In this chapter, an overview of the main integrated optical sensors will be presented with special
focus on the most relevant ones in terms of sensing performance and integration capability, and
whose feasibility for label-free biosensing has been proven. For each of them, a brief description
of its operating principle, design, fabrication and read-out resolution will be presented, and,
when reported, the biosensing dynamic range and detection limit will be summarized.
Application in clinical diagnostics, environmental monitoring, or in food and agroalimentary
industry will be included as well as a brief section describing the commercial devices already on
the market. One of the crucial aspects that will be discussed is the way towards a truly lab-on-a-
chip integration, although few examples of completely integrated platforms can be found in the
literature. Finally, an outlook of the future prospects of this technology will be discussed.
2. Biofunctionalization and immobilization strategies
The selection of an appropriate procedure to immobilize the biological element on the sensor
surface has become a critical step in the biosensor area, and enormous efforts are continuously
invested in order to optimize novel strategies according with the application. The
Page 4 of 56
Wiley - VCH
Laser & Photonics Reviews
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
5
immobilization process should not only guarantee an efficient coverage of the transducer
surface with the biomolecules, while keeping intact their properties (functionality, structure,
biological activity, affinity, specificity…), but it should also ensure their stability for storage
and regeneration. Moreover, most of the applications, such as clinical and medical diagnosis,
agroalimentary analysis, or environmental field surveillance, require well-defined surfaces with
biocompatible properties, minimizing non-specific adsorption when analyzing complex real
samples. The choice of the most effective strategy of immobilization which combines the above
considerations usually becomes the key factor which turns a sensing device into a valid and
applicable analytical tool with the required quality standards. Moreover, the bioreceptor layer
directly affects the reproducibility, selectivity and resolution of any sensor device.
A wide variety of biomolecules can be used as bioreceptors, i.e. antibodies, nucleic acid
sequences, peptides, enzymes, cell receptors and many others. The selected biomolecule is
dictated by the application and must be chosen to be highly specific for the target molecule and
stable enough to be immobilized without losing functionality. Several types of routes can be
used to biofunctionalize the sensor surface: (i) physical adsorption by direct deposition of the
biomolecule; (ii) covalent binding of the biomolecule to the surface (using a cross-linker
previously immobilized on the surface or following more complex strategies [14]); (iii) non-
covalent interactions to a previously deposited active layer, either by non-specific electrostatic
interactions or by non-covalent affinity binding (i.e. biotin-avidin systems, His-Tag system,
Protein A/G for antibodies) (iv) physical entrapment in a polymer layer. Figure 2 summarizes
the general immobilization strategies.
Physical adsorption is a simple strategy based on hydrophobic and electrostatic interactions
between the biomolecule and the surface, but it can lead to the easy desorption of the active
receptors under flow conditions and also when regeneration cocktails are applied to break the
interaction event (which usually implies high or low pH solutions, salt concentrations, organic
solvents, etc.). Moreover, issues related to reproducibility together with undesired folding of the
biomolecules onto the surface are common drawbacks of this strategy which makes it not
advisable in most of the cases even despite its simplicity.
Covalent binding can be made through one of the chemical groups of the biomolecule. It is
recommended to use a group whose blocking does not compromise the overall functionality of
the biomolecule. Amino, carboxylic or thiol groups are the preferred option to couple proteins.
For nucleic acids immobilization, it is possible to take advantage of the versatility of the DNA
synthesis which allows the incorporation of reactive groups at the end of the sequence. Specially
difficult is the attachment of antibodies in an oriented way (leaving the affinity binding sites
Page 5 of 56
Wiley - VCH
Laser & Photonics Reviews
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60