Colorimetric sensors for rapid detection of various analytes

1. Introduction

Future of sensing is based on the key factors of simplicity, cost-effectiveness and rapid response. Sensors based on colorimetric approach are significant while analyzing its ideal characteristics. Earlier sensors are mean to be a bulk and complex one, requiring different functional blocks such as transducer, processing unit, a detection unit etc. leading to a delayed sensor response. Current technology based on colorimetry is all about the miniaturization of size, cost, in-situ and without any additional instruments. A calorimetric sensor is used for instantaneous detection of analyte and shows a color change, which can be detected visually. Nanotechnology plays an important role in current sensor technology. For example, nanoparticles like Au, Ag, Cu are extensively used in visual detection owing to its optical property known as Surface PlasmonResonance (SPR). SPR is the phenomenon where collective oscillation of free electrons occurs due to resonance with incident light in visible region [1]. Plasmon resonance scattering (PRS) of Au and Ag nanoparticles have been utilized for bio-affinity sensing [2]. Generally, the detection mechanism is based on molecular interaction on the surface of the substrate which is modified or functionalized with certain functional groups or nanoparticles [1], [2], [3]. The role of nanoparticles in colorimetric sensing is reported and their results have shown that label-free assay is also possible with nanoparticles [4].

There are many challenges involved in developing an effective sensor, an ideal sensor should satisfy certain characteristics like selectivity, sensitivity, robustness, accuracy, precision, minimal error, reproducibility, linearity etc. Selectivity of a sensor is mean to be the characteristics to recognize the analyte of interest from many other interfering compounds/samples. The characteristic of a sensor to detect the analyte even at very low concentration is termed as sensitivity. It is a fact that cost may not tally with sensor characteristics. But the current technology satisfies the above-mentioned challenges. Lab-on-chip (LOC) is one among the prominent platforms on which the sensor technology is implied with high level of success [5]. It involves simple and portable devices made of polydimethylsiloxane (PDMS) being used for analyte detection by flowing liquid samples within a microchannel [5]. Microfluidics has gained wide acceptance in sensor technologies due to its low footprint and lesser user of analyte-containing reagents. Lab-on-chip technology using paper, i.e. lab-on-paper (LOP) became prominence for its low cost, rapid detection and self-sustainability. Sensor platform based on LOP for the detection of different biomolecules has already been reported by Whitesides [6]. LOP is simple, cheap and easily disposable. LOP uses cellulose paper for entrapping the molecules in a targeted site and the detection is based on the colorimetric approach. Microarray using LOP can detect different samples simultaneously.

Basically, colorimetric sensors can be classified according to the type of interaction of molecules, either chemical or biomolecules and are classified as chemical sensors and biosensors respectively. Table 1 shows a list of sensor analytes and their respective probes. In the following sections, detailed studies of different sensor systems were discussed.

Table 1. A list of sensor analytes and their respective probes.

Analyte	Probe	Reference
Chemical sensors
Volatile organic compound	Dyes	Kenneth S. Suslick et al. [31]
Volatile organic compound	Dyes	Michael C. Janzen et al. [33]
Volatile primary amine	Inkjet printing	Tamaki Soga et al. [36]
Volatile organic compound	Colloidal crystal	Tatsaro Endo et al. [34]
Organic compound	Dye	Chen Zhang et al. [35]
Odour visualization	Dye	Neal A. Rakow et al. [32]
Trinitrotoluene(TNT)	Quantum dot	Kui Zhang et al. [64]
Hg 2 + & Ag+	AuNPs	Cheng-Yan Lin et al. [49]
Thiocyanate	AuNPs	Zhang Z et al. [66]
Cu2 +	Ag nanoparticles	Nalin Ratnararthorn et al. [55]
Cu2 +	Ag nanoparticles	Yu-rong Ma et al. [53]
Cu2 +	Ag coated Au nanoparticles	Tingting Lou et al. [54]
Cu2 +	Au nanoparticles	Ruili Liu et al. [52]
Cu2 +	Au nanoparticles	Xiaorong He et al. [51]
HCL gas	Nanofibrous membrane(polyimide)	Yuan–Yuan Lv et al. [60]
H2S gas	Dye	Avijit sen et al. [61]
H2O2		Miao Xu t al [63]
Humidity	Copper nanoparticles	A. Luechinger et al. [58]
n-butyl phenol	Peptide coated SiO2	T. Kinoshita et al. [8]
NH3	Polymeric material	J. Courbat et al. [62]
Vapour sensing	Metalloporphyrin	Neal A. Rakow [32]

Biosensors
Natural amino acid	Dyes	Huo Dan-Qun et al. [16]
Protein conformational change	AuNPs	Soon woo Chah et al. [10]
Avidin	Sio2 thin film	R. Tominaga et al. [23]
Carcinoembryonic antigen	ZnFe2O4-MWCNT	Weiyan Liu et al. [24]
Antibody	Ag NP	Jian Ling et al. [25]
Heparin	TFP-Graphene oxide	Liping Cai et al. [43]
Heparin	Graphene oxide-Gold nanorods	Xiuli Fu et al. [40]
Protein	P-Phenyleneethynlylene	Oscar R. Miranda et al. [9]
Bacteria(Sphingobium yanokuyai)	RNA probe	Sivakumar et al. [27]
Histidine	Ag Nps	Haibing Lai et al. [12]
Dopamine	AuNPs	Yuanfu Zhang et al. [41]
Dopamine	Au-Ag NPs	Sivakumar et al. [42]
Bacteria	Polymer	Liron Silbert et al. [26]
DNAse I	AuNps	Weian zhao et al. [28]
Enzymatic reaction	AgNPs	Hui Wei et al. [11]
Nucleic acids	Silicon substrate	Robert Jenison et al. [29]
Biomolecules	Aptamer & Au NPs	Wei Wang et al. [13]
Antibiotic protein(kanamycin)	Aptamer&AuNPs	Kyung Mi Song et al. [14]
DNA	Aptamer & AuNps	Min sik Eom et al. [30]
Protein	Aptamer &AuNps	Jwa-Min Nam et al. [15]
L-cystein& L-Homocystein	Flourescein	Oleksander et al. [17]

2. Biosensor

Nanotechnology has its own wide range of application in the medical diagnostics field, due to its specific property of Surface Plasmon Resonance possessed by metals like Au, Ag, Cu and Pt nanoparticles [1]. For sensitive sensing of biomolecules, SPR property of nanoparticles plays a crucial role. Biosensor utilizes SPR technology for many applications like an early diagnosis of diseases such as cancer, neural disorders like Alzheimer's, Parkinson diseases etc. [3], [4]. Biosensor as the term implies that it senses biomolecules such as antigen or antibody, protein, DNA and also deals with interactions, enzyme detection, identification of microorganisms i.e., pathogen and DNA detection. The detection of analyte using colorimetric sensing is possible using surface modified nanoparticles, chemoresponsive dyes etc.

Biosensors focusing on biomimetics i.e., mimicking the nature and Pete Vukusic also studied iridescent color of butterfly wings [7]. Sensors based on biomimetics are fabricated due to the wide range acceptance of colorimetry interference. First of its kind has already been reported by Kinoshita et al. [8]using self-assembled monolayer (SAM) of polypeptides which is explained in detail in the following passages. Biosensors can be classified on the basis of detection phenomena as protein sensor, immuno sensor, pathogen sensor and DNA sensor.

2.1. Protein and amino acid sensors

Proteins are large biomolecules which are made up of amino acids. Sensing of protein molecules includes detection of amino acids, antigen-antibody interaction (immuno sensor) and also enzyme detection since all enzymes are proteins. Various amino acids or protein molecules like L-histidine, cysteine, lysozyme and methylases are detected using colorimetric approach. Few prominent examples of protein and amino acid sensors are explained below.

Vincent et al. [9] have been reported that the rapid and sensitive colorimetry sensing of bacteria using a supramolecular enzyme-nanoparticle assemblies. Protein conformational change is important in understanding the biomolecular interactions. Protein conformational changes using colorimetric sensor was reported by Richard et al. [10]. The sensor uses Au nanoparticles for visual detection. According to pH variation, folding and unfolding of protein iso 1-cytochrome (cyte) makes a clear change in the color of Au nanoparticle solution, which happens due to Surface Plasmon Resonance. In order to confirm the visible color change, UV–Visible absorption/reflectance spectroscopy was done by the authors. The protein (cyte) which is covalently bound to the gold nanoparticle upon exposure to solutions of low pH unfolds and refolds for solution having high pH value, which can be distinguished easily by the naked eye. The colorimetric sensor array for the detection of enzymatic reaction using gold nanoparticles was reported by Wang et al. [11]. The colorimetric sensor array is sensitive, selective and simple. Here enzymatic reaction such as ATP phosphorylation by calf intestine alkaline phosphatase and peptide phosphorylation by protein kinase was discussed. The enzymatic activity induces color change in gold nanoparticles. It finds applications in the screening of enzymatic inhibitors. The selective colorimetric sensing of L-histidine was developed using silver nanoparticles and Hg2 + ions reported by Li and Bian [12]. The optical property of silver nanoparticles enables the colorimetric sensing of analyte molecules. The L-cysteine modified silver nanoparticles, which are initially monodispersed in the solution, showed yellow color. Addition of Hg2 +ions helps in the binding of L-cysteine modified silver nanoparticles with amino acid. When different amino acids are added to the solution containing Hg2 +, L-cysteine modified silver nanoparticles causes the aggregation of silver nanoparticles, which shows pink color. The exception is L-histidine which shows no color change. A novel way of detecting protein molecules such as lysozyme and small molecule like ATP (Adenosine Tri Phosphate) for cell viability was performed using an aptamer-based PDMS (Polydimethylsiloxane) – Au NPs composite film [13]. The highlighting property of this biosensor is not only the detection of bio molecules, but also the catalytic efficiency of Au NPs for silver reduction. Colorimetric analysis for quantitative measurement has been done by observing the darkness density of silver enhancement.

Polydimethylsiloxane (PDMS)-Au NPs composite film with anti-target aptamer was immobilized which inhibits the reduction of silver. When the targeted aptamer get attached the catalytic property of Au NPs causes the reduction of silver with the addition of silver enhancement solution which gives a quantitative method of determination of analyte molecules as shown in Fig. 1. Here the quantitative analysis of lysozyme and ATP molecules has been done in the range of 1 × 10− 2–1 μg/mL and 1 × 10− 4 – 1x103μg/mL respectively.

Mechanism of silver enhancement is based on charge effect and spatial effect. The electrostatic repulsion between anti-target aptamer and silver lactate solution inhibits the reduction of silver where the aptamer acts as a barrier. The attachment of target molecules causes the surface charge of the aptamer to get decreased facilitating the reduction of silver. In view of spatial effect, the absence of target causes disordered immobilization of aptamer on PDMS-Au composite film. The presence of target causes the bending of the aptamer so that barrier for silver reduction gets reduced [13].

The colorimetric method of biomolecules detection using DNA and AuNPs is simple and sensitive. The distance dependent optical property and high extinction coefficient of AuNPs enable the colorimetric detection of biomolecules. Detection of antibiotic protein kanamycin using aptamer based AuNPs was reported in [14]. The presence of kanamycin causes hearing loss and toxicity to the kidneys and its detection is essential. The surface of the AuNPs is functionalized with ky2 aptamer. When NaCl is added to the solution containing kanamycin and Au NP functionalized with ky2 aptamer the color of the solution changes from red to purple. The color change occurs because of the aggregation of Au NPs by kanamycin. Further the color change was confirmed by UV–Visible absorption spectrum which shows increase in intensity at 620 nm and the intensity at 520 nm starts decreasing. This method could detect up to 25 nM. Detection of interleukin-2, a cytokine protein using colorimetric AuNPs based bio barcode DNA was reported [15]. Three types of particles namely silica micro particles, iron oxide magnetic nanoparticles and AuNPs are used where the color of the solution turns from red to blue. The detection of protein in attomolar concentration was done using this method. Colorimetric sensors using dyes can detect approximately 20 amino acids, which are present in human body. Visible sensing using chemo responsive dyes for 10 different amino acids have been studied. The platform for the detection is an array of dye having 6 × 6 sites, which will produce colorimetric response according to the amino acid to be detected, by using 36 dyes. Digital imaging of this dye before and after immersion provides the colorimetric profile, which is specific for analysis of amino acids or so called analyte. Analysis of the digital data is done by Principle Compound Analysis (PCA) and Linear Discriminant Analysis (LDA), which analyses the data statistically, and chemometricaly. The natural amino acid can be also identified from the obtained data from above analysis [16]. The colorimetric sensor for the simultaneous detection of both L-cysteine and L-homocysteine is also possible as reported previously [17]. The reaction of amino acid with fluorescence in produces thiazolidines, which induces color change for cysteine and homocysteine.

2.2. Immuno sensors

The term immuno is defined as the interaction of antibody with an antigen. Immune system of biological objects protects from pathogens and harmful diseases. Early detection of cancerous cells is possible by colorimetric immunosensors [18]. Specific antibodies functionalized to metal nanoparticlesare used for binding to biomarkers like antigens for cancer detection. After binding color change can be noted in the sensor. Similarly, different analytes like bacteria, viral particles, disease markers and even specific peptide fragment can be detected colorimetrically using nanoparticle-based immunosensors [19], [20], [21], [22].

Colorimetric detection of biomolecules could also be carried out based on thin film interference. Kinoshita et al. reported [8] that polypeptides are immobilized on the silica surface using Langmuir Blodgett Method where multilayer deposition could be achieved by this method. Interestingly, it is found that by depositing different layers of biomolecules on the silica substrate shows different colors. As thin film interference depends on the thickness of the layer and refractive index, color change can be observed [8] for different thickness as shown in Fig. 2.

Utilizing the principle of reflection and interference of scattered light is a unique technique of visual detection which is useful for molecular recognition. Biomolecules especially, proteins and small molecules can be easily detected through this method using silica substrate and the detection mechanism is thin film interference, where conventional labeling of biomolecules is not required. Multilayer made of avidin and dethiobiotin labeled BSA on silica substrate causes thin film interference [23]. Multilayers are helpful in studying the color change in thin film interference of scattering light. Color change of the thin film is proportional to the thickness and refractive index of the constructed multilayer.

The amplification of color change for detection of small molecule, i.e. by making protein multilayer that can be disassembled via molecular recognition is described by Kinoshita et al. [23]. It is also called as visible molecular affinity sensor, in which layer-by-layer multilayer of avidin and BSA modified dethiobiotin on a silica thin film disassembles while biotin is added. It is due to the fact that binding constant for biotin and avidin (1.0 × 1015 M− 1) is greater than that of dethiobiotin and avidin (5 × 1013 M− 1) thus layer thickness decreases drastically and color change is given by the interference of visible light as shown in Fig. 3.

The silica thin film is prepared by thermal oxidation method which is annealed at 960-1120 °C as shown in Fig. 4 and is modified with glycidoxyl group. This shows purple color above which multilayers of protein are constructed.

The protein layers include alternate layers of avidin and BSA modified with dethiobiotin (BSA-DSB). The increase in the layers of protein causes a color change to blue. After the construction of 8 layers a red shift occurs. When the biotin molecules are added to the multilayers, biotin avidin interaction causes the dissociation of multilayers. The change in thickness of the protein layer causes a gradual change in color causing blue shift depending on the concentration of the biotin molecule. It can be observed by Reflective VIS spectrum measurements. The spectrum of silica film with different layers was measured and red shift is observed. The possible explanation of this mechanism can be explained by using Bragg's law and Snells law and shown in Fig. 5a-b.(1) $\begin{array}{l} λ = \frac{4 h_{1}}{m} \sqrt{n_{1}^{2} - \sin^{2} α + \frac{4 h_{2}}{m} \sqrt{n_{2}^{2} - \sin^{2} α}} \\ [maximum wavelenght] m = 2 k \\ \begin{array}{c} [minimum wavelenght] & m = 2 k - 1 (k = 1, 2, 3 \dots) \end{array} \end{array}$

Reflective VIS spectra of silica thin-films with various numbers of avidin-BSA-DSB layer on their surface is shown in Fig. 6a. The dethiobiotin disassembled during different concentration of biotin added to the silica wafer. Exactly after the addition of 4.1 × 10− 3 M biotin for 36 h, the color change occurs from blue to purple. The reflective VIS spectrum shows a shift of 56.5 nm in the Fig. 6b due to the blue shift corresponds to the decrease in thickness of 7 layers.

From the data given above it is conformed, the visual detection of biotin molecule by observing the color change in the silica substrate. This method in future is expected to apply for the detection of hormones, vitamins, agrochemicals etc. An immune sensor for the detection of carcinoembryonic antigen (CEA) has been reported recently [24]. Detection is achieved based on intrinsic peroxidise activity of ZnFe2O4- Multiwall Carbon Nano Tube (ZnFe2O4MWNTs). Chitosan and porous gold deposited on filter paper paved the way for entrapping primary antibody (Ab1) which plays the role of immune sensor platform. Secondary antibodies (Ab2) were assembled on the MWNT functional group. Colorimetric sensing occurs in the presence of H2O2 oxidizing agent, where immune sensor response was quantified due to oxidization of 3,3′,5,5′-tetramethyl benzindin catalyzed by ZnFeO4-MWNT. Detection of CEA can be quantified not only by naked eye but also by digital scheme for different concentration of CEA. The label free immunoassay for antibody antigen interaction is occurred and detected using silver nanoparticles. The glass substrate immobilized with antigen, antibody is treated with the solution containing silver nanoparticles, and the AgNps are adsorbed on to the glass substrate. The visual detection is obtained by illuminating the glass slide with LED light [25].

2.3. Pathogen sensor

Most of the diseases are caused by pathogens from food contamination, unclean surroundings and insects. Adverse effects of pathogens are contagious diseases. Therefore, it is necessary to detect the pathogens as quickly as possible. Visible sensors can detect pathogens rapidly and shows the colorimetric change to identify which microorganism is detected [26].

The colorimetric sensor for the detection of bacteria has been done using self-assembled monolayer of polypeptides immobilized RNA aptamer. Conventionally, detection methods for the pathogens involve the attachment of fluorescent dyes, radioisotopes and enzymes were traditional and complex. The development of simple silica substrate along with self-assembled monolayer oligonucleotides for the attachment of bacteria was reported in [27]. Pre-colored silica chip is used as the substrate here and it is bio conjugated with poly(E-benzyloxycarbony-l-lysine) peptide (PBCL) monolayer using Langmuir Blodgett (LB) method as shown in Fig. 7.

Chip fabrication has done stage by stage, at the first stage the silane terminated polypeptide monolayer has attached to the pre-colored silicon substrate using the upward drawing LB method. Silicon wafer is pre-colored by high temperature (approximately 1060 °C for 3 h) sintering mechanism. Followed by making the PBCL monolayer coated silica surface from non-ionic to cationic by treating with HBr/CH3COOHand benzene in which the benzyloxycarbonyl group has removed and Poly l-Lysine (PLL) formed on the surface. Silicon wafer is chosen as a perfect candidate for biosensor because of its biocompatibility and ease of availability. RNA aptamer is attached to the PLL monolayer using chemical bonds where the PLL chip is dipped on RNA aptamer solution. Oligonucleotides like RNA aptamer is best suited for sensor application due to its selectivity of specific microorganism and structural stability and most of all superior and inexpensive substitutes for other ligands like antibodies. Sphingobium yanoikuyae was the specific bacteria chosen for detection. By immersing the biochip coated with RNA aptamer to the bacterial solution, binding mechanism between bacteria and the chip was studied. After air drying the chip was subjected for detailed analysis using AFM for topography and UV spectrophotometer for reflective analysis. Formation of monolayer has been understood by observing the π-A isotherm of silane terminated PBCL as shown in the Fig. 8.

From the isotherm we can see two transitions are observed, the monolayer shows a gradual rise which may be owing to flexibility shown by silane junction with the peptide terminal. It is clear from the AFM image (Fig. 9) which shows the cationic surface formed with PLL is different from the silane terminated PBCL helical rod and RNA aptamer. In addition, it is noted that the PLL monolayer does not strip off during scratching with AFM probe showing that it is attached to the silane surface not by physical adsorption but by chemical bonding.

Real-time detection of bacteria is possible when the yellow colored probe modified using RNA aptamer changes to orange due to binding of bacteria. The color change was achieved for the optimum level of bacteria, Sphingobiumconcentration. Not only by real-time monitoring but also by UV reflectance spectroscopy the detection is confirmed. As shown in Fig. 10, spectral shift is observed which indicates the presence of bacteria. Also from the reflectance spectroscopy, the shift of λmin from 463 nm to 481 nm is observed indicating the binding of bacteria. The colorimetric detection of both gram positive and gram negative bacteria has also been done using specific polymer. The bacteria secrete the enzyme, which interacts with agar-embedded nanoparticles comprising of phospholipid and polydiacetlylene polymer. The interaction causes the color of the polymer to turn from blue to red. This sensor can be used for applications like sensing in food package and for screening antibiotic resistance [26].

2.4. Nucleic acid detection

Nucleic acids such as DNA, RNA can be detected easily by using colorimetric mode of sensing. Hybridization of DNA sequences are visually detected and hence termed as colorimetric based DNA sensors. Detection of nucleic acid is essential for the early diagnosis of cancer. DNA functionalized AuNPs colorimetric sensor system has been developed for the detection of metal ions, oligonucleotides and proteins. The thermally stable, inexpensive, portable and disposable colorimetric sensor was reported in [28]. This colorimetric sensor is a paper-based sensor for the detection of DNase I and adenosine using AuNP as the probe which is either hydrophobic or hydrophilic. The paper substrate is attached with DNA-cross linked Au NP aggregates resulting in black or blue in color. When exposed to DNase I, it causes the dissociation of AuNP aggregates and shows red color within one minute. This sensor finds application in disease diagnostics, pathogen detection, quality monitoring of food and water.

Colorimetric detection of nucleic acids based on interference on optically coated silicon was reported in [29]. The polynucleotide sequence interaction can be identified to detect the specific gene (mecA gene) responsible for antibiotic resistance in S.aureus. The detection is based on interference that occurs between the substrate-thin film and the thin film-air interface. Based on the thickness of the film deposited on the substrate the wavelength of the reflected light gets varies which causes a change in the color. The silicon surface is optically coated which appears gold in white light. The capture probe is immobilized on the substrate, which is 20 nucleotide sequences. The target probe is 38 nucleotide sequence and 18 nucleotide biotin labeled oligonucleotide as the detector probe as shown in Fig. 11.

The complex was treated with HRP, which forms a thin film on the optically coated silicon substrate. The additional thickness on the substrate changes the wavelength of the reflected light and causes destructive interference where the color of the chip changes to blue. The reflectance spectra of the unreacted surface show higher reflectance in the orange red region so that the color of the substrate appears to be gold. The higher reflectance of the reacted surface in the shorter wavelength region due to the longer path length of the reflected light caused by the deposition of thin layer as shown in Fig. 12.

The polynucleotide sequence can be detected in the sub attomolar range using this detection method. This method is also sensitive that it shows color change only to methicillin resistant strain of S.aureus comparing to methicillin sensitive strain of S.aureus [29] as shown in Fig. 13.

AuNp based colorimetric sensor array for determining the binding strength of DNA has been reported in [30]. The single strand DNA functionalized with Au NPs is combined with complementary single strand DNA that causes aggregation of AuNPs resulting in the formation of duplex DNA. The dispersion of Au NPs, due to increase in temperature causes color change from blue to red. The weak binding DNA molecule changes to red color while the strong DNA binding molecule remains blue.

3. Chemical sensors

Chemical sensor is a device for detecting or sensing different chemical compounds such as volatile organic compounds, sensing gas molecules, toxic molecules and heavy metals. These chemical compounds are the effluents from the industries, which pollute the environment and causes health hazards to all living creatures. So the detection of these chemical compounds becomes necessary. Colorimetric detection techniques include array based technique, aptamer based detection, disposable paper based techniques and so on. The detection is based on intermolecular interaction of the compound present in the environment. Based on the type of the analyte detection, chemical sensor can be classified as organic compound detection, heavy metal ion detection, explosive detection and also poisonous gas detection alias gas sensing. Chemical sensors based on colorimetric detection of various analytes were discussed under different sections.