Introduction

Int J Mol Sci

ijms

International Journal of Molecular Sciences

1422-0067

Molecular Diversity Preservation International (MDPI)

PMC3100837

3100837

21614193

10.3390/ijms11125077

ijms-11-05077

Review

Colorimetric Immunoassay for Detection of Tumor Markers

Yin

Yongmei

1 * Cao

2 Xu

Yuanyuan

2 Li

Genxi

2 3 *

1 Department of Oncology, Jiangsu Province Hospital, Nanjing 210029, China 2 Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China; E-Mails: cone_zimint@yeah.net (Y.C.); ladyxuyuanyuan@gmail.com (Y.X.) 3 Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China

*Authors to whom correspondence should be addressed; E-Mails: ym.yin@hotmail.com (Y.Y.); genxili@nju.edu.cn (G.L.); Tel.: +86-25-681-360-43, +86-25-835-935-96; Fax: +86-25-837-100-40, +86-25-835-925-10.

2010

7 12 2010

11 12 5077 5094 7 11 2010 24 11 2010 25 11 2010

2010

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Tumor markers are substances, usually proteins, produced by the body in response to cancer growth, or by the cancer tissue itself. They can be detected in blood, urine, or tissue samples, and the discovery and detection of tumor markers may provide earlier diagnosis of cancer and improved therapeutic intervention. Colorimetric immunoassays for tumor marker detection have attracted considerable attention, due to their simplicity and high efficiency. The traditionally used colorimetric immunoassays for the detection of tumor markers are based on enzyme-linked immunosorbent assays, and the great achievement of nanotechnology has further opened opportunities for the development of such kind of immunoassays. This paper will summarize recent advances in the field of colorimetric immunoassays for detecting tumor markers, which is aimed to provide an overview in this field, as well as experimental guidance for the learner.

tumor markers colorimetric immunoassay nanomaterials

1. Introduction

Cancer is a leading cause of death worldwide, causing about 13% of all human deaths in 2007 (7.6 million), and deaths from cancer are projected to continue rising, with an estimated 12 million deaths projected for 2030. According to the World Health Organization, more than 30% of cancer deaths could be prevented if the cases were detected early and treated [1]. So, one key point of ultimate success for cancer treatment is the accurate diagnosis at the early stages.

Traditional methods for the diagnosis of cancer are endoscopy, biopsy and cytology specimen tests, as well as imaging/radiology tests, such as X-ray, positron emission computed tomography-computer tomography (PET-CT) and magnetic resonance imaging (MRI) [2–5]. Unfortunately, these diagnostic methods are not very powerful for the diagnosis of cancer at early stages, and some of these methods are quite time-consuming, expensive; thus not available for to a large number of people [6]. Therefore, the development of simple and rapid strategies that are specific and reliable for the diagnosis of cancer at early stages is of utter importance.

It has been known that cancer cells or other related non-tumor cells can release specific tumor markers, which are usually proteins, as tumors develop, into the circulation system in response to cancer growth. These tumor markers can be detected in blood, urine, or tissue samples, and the level of them is associated with the stage of cancer [7–10]. Alongside the development of proteomic technologies, lots of protein tumor markers have been discovered for many types of cancer (Table 1) [11–18]. Considering the high importance and potential application in the early diagnosis of cancer, detection of tumor markers has received more and more attention [19–23]. While some novel immunoassays have been developed [24–33], a variety of techniques have been employed for the development of these assays. Table 2 summarizes some examples of immunoassays which have been developed for tumor markers analysis [33–49]. Among the various detection techniques, the colorimetric method, in which the event is disclosed through a visual color change in the reaction medium, has proven the most convenient [50,51]. Thus, remarkable progress has been made on the design of colorimetric sensing systems for tumor marker detection over the years.

Large quantities of colorimetric assays for detecting tumor markers are proposed based on enzyme-linked immunosorbent assay (ELISA) [52–59]. Conventional ELISA-based colorimetric immunoassays may confront a serious sensitivity problem however, so low concentrations of tumor markers cannot be identified at the early stages of cancer. Therefore, research efforts have been focused mainly on the development of new signal amplification methods in colorimetric immunoassays. For instance, by incorporation of an enzyme-cascading step into the ELISA system, Lee et al. have reported a novel cascading ELISA, yielding detection limits of between 100 fM and 10 pM for both Prostate specific antigen (PSA) and Alpha-fetoprotein (AFP) in human serum [43]. Meanwhile, the emergence of nanotechnology is opening new horizons for highly sensitive detection of tumor markers [6,60–62]. Due to their excellent properties, many kinds of nanomaterials have been employed in colorimetric assays for detecting tumor markers [63–65].

Among the nanomaterials employed for the colorimetric detection of tumor markers, Au-NPs are the most commonly used, due to their unique optical properties. Their optical properties are strongly dependent on not only the size but also aggregation state of the particles, so smaller individual nanoparticles appear wine red while larger particles or aggregates of smaller particles range from purple to deep blue [45]. Besides, their excellent biocompatibility makes Au-NPs able to be fashioned with a wide range of biomacromolecules such as nuclear acids and enzyme-linked antibodies, greatly extending their application in the colorimetric detection of tumor markers. Therefore, large numbers of Au-NPs based colorimetric immunoassays have been developed for the detection of tumor markers, oncogenes and even tumor cells [66–70].

Apart from Au-NPs, magnetic particles (MPs) represent another exciting prospect in current analytical fields because they can be easily separated from a matrix by using a magnetic field [71–73]. Taking advantage of their unique magnetic characteristic as well as excellent biocompatibility, MPs have been widely utilized as a universal separation tool in the fabrication of colorimetric sensing systems for the detection of tumor markers [38,44,45,49,65,70].

2. Materials 2.1. ELISA-Based Colorimetric Immunoassays 2.1.1. Chemicals

96-well microtiter plates and monoclonal/polyclonal capture antibodies are used for ELISA-based colorimetric immunoassays.

Enzyme-labeled secondary antibody: hydrogen peroxidase (HRP)-labeled and alkaline phosphatase (ALP)-labeled secondary antibody have been typically used in ELISA-based colorimetric immunoassays.

Enzyme substrates: The commonly used substrate for HRP is hydrogen peroxide (H₂O₂), coupled with several hydrogen donors, such as 3,3′,5,5′-tetramethylbenzidine (TMB: Supplier: Acros Organics N.V.; Catalog No. 229280010), o-phenylenediamine dihydrochloride (OPD: Supplier: Acros Organics N.V.; Catalog No. 218480250) and 2,2′-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt (ABTS: Supplier: Roche Applied Science; Catalog No. 10102946001). The most widely used ALP substrate is p-nitrophenyl-phosphate (pNPP: Supplier: New England BioLabs; Catalog No. P0757S).

2.1.2. Buffer Reagents

Carbonate/bicarbonate coating buffer: 100 mM phosphate buffered saline (PBS), containing 28.6 mM sodium carbonate (Na₂CO₃) and 71.4 mM sodium bicarbonate (NaHCO₃), pH 9.6.

Washing buffer: 10 mM PBS, pH 7.4, containing 150 mM sodium chloride (NaCl) and 0.05% Tween-20.

Blocking buffer: PBS containing 0.1% bovine serum albumin (BSA) and 0.02% thimerosal.

All buffer reagents and other inorganic chemicals can be supplied by Sigma, Aldrich, or Fluka. All chemicals are used as received, and all aqueous solutions are prepared with doubly distilled water.

2.2. Nanomaterial-Based Colorimetric Immunoassays

Monodispersed Au-NPs with size from 2 to 250 nm can be commercially received from Ted Pella Inc. (Catalog No. 15701-15714). Au-NPs can be also synthesized in laboratory. Materials needed for the synthesis of Au-NPs are: hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O, 99.9%. Supplier: Acros Organics N.V.; Catalog No. 411070010), trisodium citrate (Citrate·3Na. Supplier: Acros Organics N.V.; Catalog No. 391970025).

MPs or functionalized MPs can be also commercially received. For example, amino-functionalized superparamagnetic microparticles (size 1–2 μm) can be obtained from Polysciences, Inc. (Catalog No. 18879); streptavidin MagneSphere® paramagnetic particles (size 0.5–1.5μm) can be purchased from Promega Corporation. (Catalog No. MD1471).

3. Methods 3.1. ELISA-Based Colorimetric Immunoassays

The most frequently used ELISA method in colorimetric immunoassays for tumor marker detection is the two antibody “sandwich” ELISA, which measures the amount of antigen between two layers of antibodies [52]. In this assay, a capture antibody is firstly immobilized onto a solid support. The tumor marker, which is present in a biological sample or standard mixture, is then bounded and concentrated onto the support surface during incubation. After that, the solid support is incubated with a solution containing the detection antibody, thus the detection antibody will also bind with the tumor marker. Since the detection antibody is also tagged with certain enzymes, the color change caused by the enzyme-catalytic reaction can be thus used to quantify the amount of target tumor marker. The experimental details are described below, mainly with a slightly modified procedure recommended by Abcam [74].

3.1.1. Coating with Capture Antibody

Coat the wells of a microtiter plate with the capture antibody at a concentration of 1–10 μg/mL in carbonate/bicarbonate coating buffer. Then cover the microtiter plate with an adhesive plastic. After incubation overnight at 4 °C, remove the coating solution and wash the plate at least twice by filling the wells with 200 μL of washing buffer. The solution in the wells can be removed by flicking the plate over a sink, and the remaining drops in the wells can be removed by patting the plate on a paper towel.

3.1.2. Blocking and Adding Samples

200 μL of blocking buffer containing 5% non fat dry milk is firstly added into each well to block the remaining protein-binding sites. After that, the plate is covered with an adhesive plastic and incubated with the blocking buffer for at least 1–2 h at room temperature or overnight at 4 °C. Then add 100 μL of appropriately diluted samples into each well, and incubate the plate with the sample solution for 90 min at 37 °C. After incubation, remove the samples and wash the plate twice by filling the wells with 200 μL of washing buffer.

3.1.3. Incubation with Detection Antibody

Dilute the detection antibody appropriately with 0.1 M bicarbonate buffer (pH 9.2). Then, add 100 μL of the buffer solution to each well. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. After that, wash the plate four times with washing buffer.

3.1.4. Colorimetric Detection

Although many different types of enzymes have been used in ELISA, HRP and ALP are the two most widely used enzymes [43]. The substrate for HRP is H₂O₂, and the cleavage of H₂O₂ is usually coupled with the oxidation of a hydrogen donor which may change the color of the test solution during the reaction, so hydrogen donors should also be involved in the reaction. These hydrogen donors, usually TMB, OPD and ABTS, are light sensitive, so they need to be stored in the dark. Furthermore, OPD is considered hazardous (potential carcinogens), so it always needs to be handled with care. In a typical experiment, H₂O₂ and hydrogen donors are added into each well and incubated for 15–30 min. After that, equal volume of stopping solution (2 M H₂SO₄) is added to stop the reaction. Finally, the absorbance of the product of TMB oxidation is measured at the wavelength of 450 nm, while OPD at 492 nm; ABTS at 416 nm.

The most widely used ALP substrate is pNPP. The yellow color of the product nitrophenol can be measured at 405 nm after 15–30 min incubation at room temperature, and this reaction can be stopped by adding equal volume of 0.75 M NaOH.

3.2. Nanomaterial-based Colorimetric Immunoassays

The unique properties of nanomaterials may offer a wide range of opportunities in colorimetric immunoassays of tumor markers. In the following section, we provide experimental details for the synthesis, surface modification and utilization of nanomaterials in the fabrication of colorimetric sensing systems for the detection of tumor markers.

3.2.1. Synthesis and Modification of Nanomaterials 3.2.1.1. Synthesis of 13 nm Au-NPs

All glassware used in the synthesis procedures should be firstly immersed in freshly prepared aqua regia (HNO₃: HCl = 1:3) for 30 min, then washed with water and dried before use. Au-NPs are synthesized by reducing HAuCl₄ with Citrate·3Na. Briefly, a 100 mL aqueous solution of 0.01% (w/v) HAuCl₄ is added into a round-bottom flask and stirred to boil. Then, 3.5 mL 1% trisodium citrate is added rapidly into the boiling solution, the color of which changes from colorless to wine red after boiling for another 15 min with vigorous stirring. The size of the nanoparticles is 12.5 ±2.3 nm, which can be determined by transmission electron microscope (TEM). The concentration of Au-NPs is 3.5 nM, which can be calculated from the quantity of starting material (HAuCl₄) and the size of Au-NPs at a wavelength of 520 nm [45].

3.2.1.2. Modification of Au-NPs

The main technique reported for the modification of Au-NPs surfaces is based on Au-S covalent bond formation between the modified molecules and the gold atoms on the particle surface [71], which makes use of sulphur-containing ligands, such as thiol, disulphide and thiolester.

3.2.1.2.1. Modification of Au-NPs with Thiol-oligonucleotides

The above prepared Au-NPs solution is firstly condensed by centrifugation at 12,000 rpm for 20 min at 4 °C to a 1 mL Au-NPs solution, while 800 μL supernatant is removed; the remaining 200 μL is redispersed. After condensation, 800 μL Au-NPs solution is mixed with 200 μL, 5 μM 5′-thiol-oligonucleotides at room temperature. After 24 h duration in darkness, the mixture is centrifuged twice, each for 20 min at 12,000 rpm to remove the excess thiol-oligonucleotides. After centrifugation, the precipitate is washed with 4 mM Citrate·3Na. The Au-NPs, modified with thiol-oligonucleotides, are stored at 4 °C when not in use. To determine the number of thiol-oligonucleotides modified on each Au-NP, 2-mercaptoethanol (1.0 M, 10 μL), which has a smaller size than oligonucleotides and is easier to be modified on the surface of Au-NPs through Au-S bond, is used to replace thiol-oligonucleotides from the surface of Au-NPs (11 nM, 990 μL). The amount of replaced thiol-oligonucleotides in the supernatant liquor can be thus used to calculate the quantity on each Au-NP [75].

3.2.1.2.2. Modification of Au-NPs with Antibodies

The methods to modify Au-NPs with antibody include adsorption [76], N-hydroxysuccinimide (NHS) ester chemistry [77], and oligonucleotide-directed immobilization [78]. Among them, the direct adsorption method is the simplest one, due to the fact that neutral or weakly negative-charged proteins can be adsorbed on the surface of Au-NPs easily and stably through coordinate-covalent bond [79]. The preparation of Au-NPs modified with antibodies through direct adsorption is as follows [45]: 20 μL of protein solution (1 mg/mL) is first added into 5 mL of the above prepared Au-NPs solution, and the pH is appropriately adjusted by adding 1 M NaOH solution. Then the mixed solution is incubated for 30 min at 37 °C with gentle shaking, and centrifuged to 1 mL (12,000–13,000 rpm, 20–30 min, 4 °C). After that, 100 μL of 10% (w/v) BSA solution, used as non-specific blocker, is added into the solution to passivate the surface of Au-NPs. After 30 min, the mixed solution is again centrifuged (12,000–13,000 rpm, 20–30 min, 4 °C). The supernatant is subsequently decanted, and the precipitate is rinsed with 1 mL of PBS buffer. The centrifuging/rinsing procedure should be repeated three times to remove the unbounded antibodies and BSA.

3.2.1.3. Modification of MPs

MPs may provide an efficient tool of separating target analytes from the liquid suspension; however, unmodified MPs have some limitations in the applications. Therefore, MPs surfaces should first of all be modified with some specific linking groups for further biomolecules binding, including: (1) Active chemical groups (e.g., carboxyl and amino groups), which can covalently bind biomolecules in the presence of specific cross-linking reagents; (2) streptavidin/avidin ligands, which can be specifically attached to biotinylated biomolecules [71–73]. Taking into account that amino- or streptavidin-functionalized MPs are commercially available, the following is only a description of the modification by antibodies for these pre-functionalized MPs.

3.2.1.3.1. Modification of Amino-Functionalized MPs

Amino-functionalized MPs can be modified with antibodies by using glutaraldehyde-amine coupling chemistry [33]. Briefly, amino-functionalized MPs (0.05 mM) in 1 mg mL⁻¹ of ethylenediaminetetraacetic acid (EDTA) solution (5 mL) are firstly washed with 5% (v/v) pyridine washing buffer (10 mL). Then, MPs are separated magnetically. This process should be repeated three times. After that, the MPs are activated by glutaraldehyde in pyridine washing buffer (5 mL, 5%) for 3 h at room temperature, separated magnetically twice, and resuspended in pyridine washing buffer (10 mL). Finally, antibody dissolved in pyridine wash buffer (1 mL at 1 mg mL⁻¹) is added to the MPs solution. After 10 h duration at room temperature, 1 mg BSA is added to the MPs solution. After another 10 h at room temperature, the MPs are washed twice and then resuspended in pyridine washing buffer (5 mL). Subsequently, glycine solution (3 mL, 1 M at pH 8.0) is added into the above solution to quench all of the unreacted aldehyde groups, and the resulting solution is stirred for 30 min. After magnetic separation three times and the washing step, MPs modified with antibody (Ab-MPs) are resuspended in PBS solution.

3.2.1.3.2. Modification of Streptavidin-Functionalized MPs

Streptavidin-functionalized MPs are firstly washed three times with washing buffer (75 mM NaCl, 7.5 mM Citrate·3Na, pH 7.0) prior to use. Then they are diluted to 0.1 mg mL⁻¹ with PBS, pH 7.4. After that, a solution of biotinylated antibody is added to the MPs solution at a ratio of 100 μg protein to 1 mg MPs, and the mixed solution is incubated for 20 min at room temperature. Finally, the Ab-MPs are thoroughly washed three times with pH 7.4 PBS [45].

3.2.2. Magnetic Collection and Separation of Target Tumor Markers

MPs modified with capture antibodies can be used for fast target protein collection and separation. In a typical experiment, 50 μL of capture antibody-modified MPs (0.2 mg mL⁻¹) are first added to a 200 μL eppendorf tube. Then, 50 μL of assay buffer containing various concentrations of target tumor markers are added. After incubation for 30 min at 37 °C with gentle shaking, the magnetic beads are magnetically collected and twice rinsed with washing buffer [70].

3.2.3. Application of Au-NPs in Colorimetric Immunoassays of Tumor Markers

Basically, two colorimetric procedures by making use of Au-NPs have been proposed for tumor marker quantification: (a) Analysis of the state change of Au-NPs in solution, including the homogenous growth and aggregation triggered by a biological process; (b) analysis of the activity of enzymes that attached to Au-NPs, in which case Au-NPs act as enzyme carriers and signal enhancers.

3.2.3.1. Colorimetric Assay Using Homogenous Growth of Au-NPs

10 mL solution for the growth of Au-NPs is first prepared in a 20 mL vial, consisting of final effective concentrations of 0.25 mM HAuCl₄ and 0.1 M cetyltrimethylammonium bromide (CTAB). Before use, the solution is gently mixed with ascorbic acid (AA) solution with a final AA concentration of 10 mM and stirred. Seperately, Au-NPs/tumor marker complexes are first separated through magnetic separation and washed with 0.01 M PBS buffer (pH 7.4) three times to remove the excess of free Au-NPs. Then, Au-NPs are dissociated from the complexes by using 50 mM NaCl/1 M NaOH elution solution. Finally, the upper aqueous solution containing the dissociated Au-NPs is transferred to a 1 mL cuvette, followed by addition of the above previously prepared 1 ml solution for the growth of Au-NPs. The growth of Au-NPs and the color change of the solution are then recorded [38].

3.2.3.2. Colorimetric Assay Using Aggregation of Au-NPs

In this section, we introduce the experimental details of two typical assays. One is based on single chain fragment variable recombinant antibody (scFv)-functionalized Au-NPs, while the other is colorimetric bio-barcode assay.

3.2.3.2.1. scFv-Functionalized Au-NPs Based Assay

Firstly, scFv-functionalized Au-NPs are prepared as described in Section 3.2.1.2.2. Then, a range of concentrations of target protein are added to the solution of the scFv-functionalized Au-NPs while stirring, thus the scFv-functionalized Au-NPs show visible color change due to the aggregation of the nanoparticles caused by the interaction of scFv with the target protein. After 30 min, the developing reaction can be recorded by using a UV-visible spectrophotometer [69].

3.2.3.2.2. Colorimetric Bio-Barcode Assay

In a typical experiment, two types of probes are prepared. The first is a magnetic probe, i.e., MP modified with monoclonal antibodies for target tumor marker, prepared as described in Section 3.2.1.3.1. The second is a barcode probe, i.e., a porous silica particle modified with the monoclonal antibodies and barcode DNA complements. To prepare the barcode probe, amino-functionalized porous silica microparticles are firstly linked to antibodies using glutaraldehyde-amine coupling chemistry. Then, 3′-amino-functionalized barcode DNA complements (1 mL at 100 μM) are added to the solution containing the antibody modified silica particles. After incubation overnight at room temperature with gentle shaking, the solution is centrifuged twice, and the pellets are resuspended in 1 mL of 0.2 M ethanolamine. After 30 min duration at room temperature, centrifugation is performed again to remove the supernatant, and a 10% BSA solution is subsequently added, followed by repeating the centrifugation steps twice. Finally, the resulting pellets are resuspended in 1 mL of 0.15 M PBS solution, which is further mixed with 30 μL 100 μM barcode DNA solution and incubated for 1 h at room temperature.

In the tumor marker detection assay, 15 μL of magnetic probe solution (1.5 × 10⁹ beads/mL) is first added to 20 μL of tumor marker solution, followed by the addition of 15 μL of barcode probe solution (1 × 10⁹ beads/mL). The mixed solution is incubated at 37 °C for 50 min on an orbital shaker. Then, the solution is magnetically separated as described in Section 3.2.2, and the magnetically separated complexes are washed with 0.15 M PBS solution three times. After that, 50 µL of NANOpure water (18 MΩ) is added to the complexes, and the solution is kept on a rocking shaker at 70 °C for 10 min to release the barcode DNA. Finally, the supernatant containing free barcode DNA strands is collected for barcode DNA detection. To detect the barcode DNA, DNA-functionalized Au-NPs (prepared as described in Section 3.2.1.2.1) are added to the barcode DNA in 0.15 M PBS solution. Due to the hybridization between the barcode DNA and its complements that are modified on Au-NPs surfaces, Au-NPs aggregate and show visible color change. And, the mixed solution, after being maintained at room temperature for 2 h, can be transferred into a 100-μL quartz cuvette to measure the extinction coefficients by using a UV-vis spectrophotometer [29,68].

3.2.3.3. Colorimetric Assay using Enzymes Labeled Au-NPs

Due to the high loading of signaling molecules on each nanoparticle, greatly enhanced sensitivity for the detection of tumor markers can be achieved by using enzyme labeled Au-NPs [70]. In such colorimetric assays, Au-NPs play a role of enzyme carriers and signal amplifiers, and the detectable signals come from the catalytic reaction of the enzymes that are loaded onto the nanoparticle surfaces. The commonly used enzymes and substrates are the same as those used in ELISA-based colorimetric assays.

4. Results and Discussion

As an example, one recent work in the authors’ laboratory on colorimetric multiplexed immunoassays is introduced [45]. Figure 1 illustrates the principle of the assay for sequential detection of tumor markers. Briefly, biotin-anti-CEA conjugated MPs are first added to the test solution that contains CEA, AFP and some nonspecific proteins. After magnetic separation, CEA antigen which is captured by biotin-anti-CEA, immobilized on the surface of MPs, is thus collected. Therefore, if Au-NPs loaded with HRP-anti-CEA are added to the collected CEA antigen solution, HRP-anti-CEA loaded on the surface of Au-NPs may capture the CEA antigen conjugated on the surface of MPs. Consequently, when TMB and H₂O₂ are added to the test solution, its color turns blue. Alternatively, the detection of AFP antigen in the test solution can be carried out by repeating the above procedure, using biotin-anti-AFP conjugated MPs and Au-NPs loaded with HRP-anti-AFP.

The multiplexed immunoassay is very simple and easily operated, and the results can be seen by the naked eye (Figure 2). Meanwhile, the absorbance at 370 nm or 490 nm of the test solutions can be separately used to determine the amount of tumor markers more accurately. Under optical conditions, the immunoassay can provide sequential detection of CEA and AFP with detection limits of 0.02 ng/mL (≈0.1 pM), which can be satisfactory for clinical applications.

Colorimetric assay is very simple and easily operated, without requiring the expensive instruments needed in the optical immunoassay systems, such as charge-coupled device (CCD) camera and multi-channel injection valves fixed to luminescence analyzers. In fact, electrochemical assay is also very simple and relatively inexpensive, thus some electrochemical methods have also been proposed for the detection of cancer. However, the current amplification strategy for electrochemical signal often involves multiple steps of deposition and stripping, making the experimental process complex; while colorimetric approaches do not require complicated experimental steps, and results can be seen with the human naked eye. Therefore, colorimetric immunoassay for the detection of tumor markers has received more and more interest. Meanwhile, nanotechnology has greatly promoted the development of colorimetric immunoassays. Firstly, MPs may provide a rapid and safe approach to separate the target from the other species, preventing the interference of non-specific proteins in immunoassays. Secondly, the unique optical properties of Au-NPs can be harnessed to realize the transduction of the presence of target tumor markers to easily detectable signals, simplifying the designed detection system. Thirdly, the use of nanomaterials has enabled detection of tumor markers with greater sensitivity and accuracy. Therefore, colorimetric immunoassays have made prominent progress in the detection of tumor markers and may play an important role in the early diagnosis of cancer.

Colorimetric immunoassays have many desirable merits, thus are rapidly developing. Tremendous opportunities and challenges exist in the application of colorimetric methods for tumor marker detection.

Firstly, colorimetric immunoassays for tumor marker detection are usually proposed by using antibodies; however the availability of antibodies with high affinity and low cross-reactivity are very limited. Fortunately, aptamers—nucleic acid molecules that can selectively bind to low molecular weight organic or inorganic substrates or to macromolecules such as proteins—can be considered as attractive alternatives to antibodies [45]. In fact, compared with antibodies, aptamers may possess many advantages, such as relatively simple and inexpensive synthesis, tolerance to internal labeling, high stability, and stronger and more selective affinity for protein targets. Therefore, several aptamers have been employed for tumor marker detection [80,81].

Secondly, nanomaterials used in colorimetric methods for sensing tumor markers are still narrowly restricted to Au-NPs and MPs. Recently, some novel types of nanomaterials were fabricated and used in colorimetric biosensors for various analytes [82–84], so more nanomaterials will be applied in the development of colorimetric immunoassays for tumor marker detection, simplifying the assay methodology and lowering the detection limit as far as possible.

Finally, the measurement of a single tumor marker is usually not sufficient for diagnosis purposes, because most cancers have more than one marker associated with their incidence [85], thus multiplexed immunoassays that can quantitatively measure the concentrations of multiple tumor markers in a single assay have received more and more interest [25,46]. Compared with the traditional parallel single-analyte immunoassay, multiplexed immunoassays have the advantage of simplified analytical procedure, shortened analysis time, decreased sample consumption, increased test throughput, reduced costs, and improved diagnostic accuracy [45]. Consequently, future advances in colorimetric immunoassays will depend on the development of simple and efficient sensing mechanisms for multiplexed tumor marker examinations.

References 1.

World Healther Organization

Cancer

Available online: http://www.who.int/mediacentre/factsheets/fs297/en/ (accessed on 25 November 2007).

Reid

Weinstein

Lewin

Haggitt

van Deventer

DenBesten

Rubin

Endoscopic biopsy can detect high-grade dysplasia or early adenocarcinoma in Barrett’s esophagus without grossly recognizable neoplastic lesions

Gastroenterology 1988 6 81 90

3335302

Wahl

Quint

Greenough

Meyer

White

Orringer

Staging of mediastinal non-small cell lung cancer with FDG PET, CT, and fusion images: Preliminary prospective evaluation

Radiology 1994 191 371 377

8153308

Beyer

Townsend

Brun

Kinahan

Charron

Roddy

Jerin

Young

Byars

Nutt

A combined PET/CT scanner for clinical oncology

J. Nucl. Med 2000 41 1369 1379

10945530

Kircher

Mahmood

King

Weissleder

Josephson

A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation

Cancer Res 2003 63 8122 8125

14678964

Choi

Kwak

Park

Nanotechnology for early cancer detection

Sensors 2010 10 428 455

Safi

Kohler

Rottinger

Beger

The value of the tumor marker CA15-3 in diagnosing and monitoring breast cancer

Cancer 1991 68 574 582

2065278

Harris

Fritsche

Mennel

Norton

Ravdin

Taube

Somerfield

Hayes

Bast

American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer

J. Clin. Oncol 2007 25 5287 5312

17954709

Kumar

Mohan

Guleria

Biomarkers in cancer screening, research and detection: Present and future: A review

Biomarkers 2006 11 385 405

16966157

10.

Hanash

Pitteri

Faca

Mining the plasma proteome for cancer biomarkers

Nature 2008 452 571 579

18385731

11.

Chen

Sung

Serum alpha-fetoprotein in hepatocellular carcinoma

Cancer 1977 40 779 783

70268

12.

Canney

Moore

Wilkinson

James

Ovarian cancer antigen CA125: A prospective clinical assessment of its role as a tumour marker

Br. J. Cancer 1984 50 765 769

6208925

13.

Benchimol

Fuks

Jothy

Beauchemin

Shirota

Stanners

Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule

Cell 1989 57 327 334

2702691

14.

Osborne

Yochmowitz

Knight

3rd McGuire

The value of estrogen and progesterone receptors in the treatment of breast cancer

Cancer 1980 46 2884 2888

7448733

15.

Jacobsen

Alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) in testicular germ cell tumours: A prospective immunohistochemical study

Acta Pathol. Microbiol. Immunol. Scand. A 1983 91 165 176

6190350

16.

Horton

Her2 and Trastuzumab in breast cancer

Cancer Control 2001 8 103 110

11176040

17.

Lilja

Ulmert

Vickers

Prostate-specific antigen and prostate cancer: Prediction, detection and monitoring

Nat. Rev. Cancer 2008 8 497 511

18563191

18.

Stamey

Yang

Hay

McNeal

Freiha

Redwine

Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate

N. Engl. J. Med 1987 317 909 916

2442609

19.

Catalona

Smith

Ratliff

Dodds

Coplen

Yuan

Petros

Andriole

Measurement of prostate-specific antigen in serum as a screening test for prostate cancer

N. Engl. J. Med 1991 324 1156 1161

1707140

20.

Dhanasekaran

Barrette

Ghosh

Shah

Varambally

Kurachi

Pienta

Rubin

Chinnaiyan

Delineation of prognostic biomarkers in prostate cancer

Nature 2001 412 822 826

11518967

21.

Bates

Clinical applications of serum tumor markers

Ann. Int. Med 1991 115 623 638

1716430

22.

Carter

Presta

Gorman

Ridgway

Henner

Wong

Rowland

Kotts

Carver

Shepard

Humanization of an anti-p185HER2 antibody for human cancer therapy

Proc. Natl. Acad. Sci. USA 1992 89 4285 4289

1350088

23.

Bast

Bates

Bredt

Desch

Fritsche

Fues

Hayes

Kemeny

Kragen

Jessup

Locker

Macdonald

Mennel

Norton

Ravdin

Smith

Taube

Winn

Clinical practice guidelines for the use of tumor markers in breast and colorectal cancer

J. Clin. Oncol 1996 14 2843 2877

8874347

24.

Sok

Clarizia

Farris

McDonald

Novel fluoroimmunoassay for ovarian cancer biomarker CA-125

Anal. Bioanal. Chem 2009 393 1521 1523

19145430

25.

Wilson

Nie

Multiplex measurement of seven tumor markers using an electrochemical protein chip

Anal. Chem 2006 78 6476 6483

16970323

26.

Curreli

Lin

Lei

Ishikawa

Datar

Cote

Thompson

Zhou

Complementary detection of prostate-specific antigen using In₂O₃ nanowires and carbon nanotubes

J. Am. Chem. Soc 2005 127 12484 12485

16144384

27.

Nam

Thaxton

Mirkin

Nanoparticle-based bio–barcodes for the ultrasensitive detection of proteins

Science 2003 301 1884 1886

14512622

28.

Zheng

Patolsky

Cui

Wang

Lieber

Multiplexed electrical detection of cancer markers with nanowire sensor arrays

Nat. Biotechnol 2005 23 1294 1301

16170313

29.

Nam

Wise

Groves

Colorimetric bio-barcode amplification assay for cytokines

Anal. Chem 2005 77 6985 6988

16255599

30.

Zhao

Sun

Zhu

Encoded porous beads for label-free multiplex detection of tumor markers

Adv. Mater 2008 20 1 4

31.

Liu

Dai

Austin

Coutts

Knowles

Zou

Chen

Huo

A One-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering

J. Am. Chem. Soc 2008 130 2780 2782

18257576

32.

Stoeva

Lee

Smith

Rosen

Mirkin

Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes

J. Am. Chem. Soc 2006 128 8378 8379

16802785

33.

Nam

Lee

Mirkin

A fluorophore-based bio-barcode amplification assay for proteins

Small 2006 2 103 108

17193564

34.

Datar

Hansen

Thundat

Cote

Majumdar

Bioassay of prostate-specific antigen (PSA) using microcantilevers

Nat. Biotechnol 2001 19 856 860

11533645

35.

Mani

Chikkaveeraiah

Patel

Gutkind

Rusling

Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification

ACS Nano 2009 3 585 594

19216571

36.

Munge

Patel

Jensen

Bhirde

Gong

Kim

Gillespie

Gutkind

Papadimitrakopoulos

Rusling

Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers

J. Am. Chem. Soc 2006 128 11199 11205

16925438

37.

Grubisha

Lipert

Park

Driskell

Porter

Femtomolar detection of prostate-specific antigen: An immunoassay based on surface-enhanced Raman scattering and immunogold labels

Anal. Chem 2003 75 5936 5943

14588035

38.

Cao

Lee

Sim

Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker

Biosens. Bioelectron 2009 24 1292 1297

18783934

39.

Gao

Pan

Cui

Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles

Anal. Chem 2006 78 1104 1106

16478100

40.

Lee

Kim

Park

Yeo

Mass spectrometry signal amplification method for attomolar detection of antigens using small-molecule-tagged gold microparticles

Angew. Chem. Int. Ed 2008 47 9518 9521

41.

Yuan

Chai

Chen

New antibody immobilization strategy based on gold nanoparticles and azure I/multi-walled carbon nanotube composite membranes for an amperometric enzyme immunosensor

J. Phys. Chem. C 2007 111 8443 8450

42.

Zhou

Zhang

Multilayers enzyme-coated carbon nanotubes as biolabel for ultrasensitive chemiluminescence immunoassay of cancer biomarker

Biosens. Bioelectron 2009 24 2961 2966

19345084

43.

Lee

Jeong

Kang

Chung

Cascade enzyme-linked immunosorbent assay (CELISA)

Biosens. Bioelectron 2009 25 332 337

19665363

44.

Zhou

Zhu

Guo

Chen

Yang

Wang

Amplified detection of protein cancer biomarkers using DNAzyme functionalized nanoprobes

Chem. Commun 2009 44 6845 6847

45.

Wang

Cao

Colorimetric multiplexed immunoassay for sequential detection of tumor markers

Biosens. Bioelectron 2009 25 532 536

19726177

46.

Liu

Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers

Anal. Chem 2006 78 6999 7005

17007526

47.

Yuan

Chai

Tang

Chai

A novel amperometric immunosensor based on layer-by-layer assembly of gold nanoparticlesmulti-walled carbon nanotubes-thionine multilayer films on polyelectrolyte surface

Anal. Chim. Acta 2007 603 205 213

17963841

48.

Chon

Lee

Son

Choo

Highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced raman scattering of hollow gold nanosperes

Anal. Chem 2009 81 3029 3034

19301845

49.

Liu

Jia

Huang

Lou

Yao

Jin

Zhao

Xiang

Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes

Analyst 2010 135 327 331

20098766

50.

Knecht

Sethi

Bio-inspired colorimetric detection of Hg²⁺ and Pb²⁺ heavy metal ions using Au nanoparticles

Anal. Bioanal. Chem 2009 394 33 46

19189085

51.

Ohno

CIE fundamentals for color measurements

IS&T NIP16 Intl. Conf. on Digital Printing Technologies

Vancouver, Canada

2000 540 545

52.

Zangar

Daly

White

ELISA microarray technology as a high-throught system for cancer biomarker validation

Expert Rev. Proteomics 2006 3 37 44

16445349

53.

Lequin

Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)

Clin. Chem 2005 51 2415 2418

16179424

54.

Leng

McElhaney

Walston

Xie

Fedarko

Kuchel

Elisa and multiplex technologies for cytokine measurement in inflammation and aging research

J. Gerontol. A Biol. Sci. Med. Sci 2008 63 879 884

18772478

55.

Cook

Bustamam

Brotherick

Shenton

Self

Lectin ELISA for the c-erb-B2 tumor marker protein p185 in patients with breast cancer and controls

Clin. Chem 1999 45 292 295

9931057

56.

Zehentner

Persing

Deme

Toure

Hawes

Brooks

Feng

Hayes

Critichlow

Houghton

Kiviat

Mammaglobin as a novel breast cancer biomarker: Multigene reverse transcription-PCR assay and sandwich ELISA

Clin. Chem 2004 50 2069 2076

15375015

57.

Prasad

Chaube

Shrivastav

Kumari

Development of colorimetric enzyme-linked immunosorbent assay for human chorionic gonadotropin

J. Immunoassay Immunochem 2006 27 15 30

16450866

58.

Ambrosi

Airò

Merkoçi

Enhanced gold nanoparticle based ELISA for a breast cancer biomarker

Anal. Chem 2010 82 1151 1156

20043655

59.

Liu

Wang

Huang

Yang

Liu

Yang

Microchip-based ELISA strategy for the detection of low-level disease biomarker in serum

Anal. Chim. Acta 2009 650 77 82

19720177

60.

Ferrari

Cancer nanotechnology: Opportunities and challenges

Nat. Rev. Cancer 2005 5 161 171

15738981

61.

Chen

Jiang

Zhang

Liu

Kong

Protein chips and nanomaterials for application in tumor marker immunoassays

Biosens. Bioelectron 2009 24 3399 3411

19398322

62.

Gomez-Hens

Fernandez-Romero

Aguilar-Caballos

Control of tumor markers using nanotechnology

Mini-Rev. Med. Chem 2009 9 1064 1074

19689403

63.

Kim

Daniel

Mirkin

Microarray-based multiplexed scanometric immunoassay for protein cancer markers using gold nanoparticle probes

Anal. Chem 2009 81 9183 9187

19874062

64.

Ling

Huang

Visual sandwich immunoassay system on the basis of plasmon resonance scattering signals of silver nanoparticles

Anal. Chem 2009 81 1707 1714

19173573

65.

Piao

Lee

Kim

Hyeon

Kim

High performance immunoassay using immobilized enzyme in nanoporous carbon

Analyst 2009 134 926 932

19381386

66.

Arumugam

Senapati

Singh

Arbneshi

Khan

Ray

Multifunctional oval-shaped gold nanoparticle-based selective detection of breast cancer cells using simple colorimetric and highly sensitive two photon scattering assay

ACS Nano 2010 4 1739 1749

20155973

67.

Tan

Liu

Thomsen

Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions

Anal. Chem 2010 82 2759 2765

20199029

68.

Nam

Jang

Groves

Detection of proteins using a colorimetric bio-barcode assay

Nat. Protoc 2007 2 1438 1444

17545980

69.

Liu

Mernaugh

Zeng

Single chain fragment variable recombinant antibody functionalized gold nanoparticles for a highly sensitive colorimetric immunoassay

Biosens. Bioelectron 2009 24 2853 2857

19327975

70.

Liu

Jia

Jin

Lou

Yao

Xiang

Zhao

Novel colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen

Talanta 2010 81 1625 1629

20441949

71.

de Dios

Díaz-García

Multifunctional nanoparticles: Analytical prospects

Anal. Chim. Acta 2010 666 1 22

20433959

72.

Stanciu

Won

Ganesana

Andreescu

Magnetic Particle-Based Hybrid Platforms for Bioanalytical Sensors

Sensors 2009 9 2976 2999

73.

Hsing

Zhao

Micro- and nano-magnetic particles for applications in biosensing

Electroanalysis 2007 19 755 768

74.

Sandwich ELISA Protocol

General procedure and tips for Sandwich ELISA including details of how to find matched pair antibodies

Abcam

Hong Kong, China

Available at: http://www.abcam.cn/index.html?pageconfig=resource&rid=11422 (accessed on 25 November 2010).

75.

Huang

Cao

Tan

Chang

Aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors

Anal. Chem 2005 77 5735 5741

16131089

76.

Faulk

Taylor

An immunocolloid method for the electron microscope

Immunochemistry 1971 8 1081 1083

4110101

77.

Qian

Peng

Ansari

Yin-Goen

Chen

Shin

Yang

Young

Wang

Nie

In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags

Nat. Biotechnol 2008 26 83 90

18157119

78.

Niemeyer

Ceyhan

DNA-directed functionalization of colloidal gold with proteins

Angew. Chem. Int. Ed 2001 40 3685 3688

79.

Chen

Tsai

Huang

Chang

Cheng

Chou

Chen

Wang

Shiau

Methotrexate conjugated to gold nanoparticles inhibits tumor growth in a syngeneic lung tumor model

Mol. Pharm 2007 4 713 722

17708653

80.

Ferreira

Papamichael

Guilbault

Schwarzacher

Gariepy

Missailidis

DNA aptamers against the MUC1 tumour marker: Design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours

Anal. Bioanal. Chem 2008 390 1039 1050

17694298

81.

Lupold

Hicke

Lin

Coffey

Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen

Cancer Res 2002 62 4029 4033

12124337

82.

Kim

Seo

Jung

Functionalized monolayers on mesoporous silica and on titania nanoparticles for mercuric sensing

Analyst 2010 135 149 156

20024195

83.

Song

Wang

Zhao

Ren

Label-free colorimetric detection of single nucleotide polymorphism by using single-walled carbon nanotube intrinsic peroxidase-like activity

Chem. Eur. J 2010 16 3617 3621

84.

Chen

Luo

Lin

Chen

Sheng

Chen

Liu

Cai

Label-free colorimetric assay for biological thiols based on ssDNA/silver nanoparticle system by salt amplification

Analyst 2010 135 1066 1069

20405067

85.

Yan

Biomedical and clinical applications of immunoassays and immunosensors for tumor markers

Trends Anal. Chem 2007 26 679 688

Figure 1.

Figure 2.

Colorimetric detection of CEA and AFP antigens. Only in the presence of antigens which are cognate with the antibodies loaded on the surfaces of MPs and Au-NPs, can the immunocomplexes be formed, and the characteristic blue and yellow colors of catalytic products be observed. Reprinted with permission from Ref. [45]. Copyright 2009 Elsevier.

Table 1.

Some tumor markers currently in use.

Tumor Markers	Related Cancers	Usual Sample	Refs.
AFP (Alpha-fetoprotein)	Liver, germ cell cancer of ovaries or testes	Blood	[11]
CA 15-3 (Cancer antigen 15-3)	Breast	Blood	[7]
CA-125 (Cancer antigen 125)	Ovarian	Blood	[12]
CEA (Carcinoembryonic antigen)	Colorectal, breast, thyroid et al.	Blood	[13]
Estrogen receptors	Breast	Tissue	[14]
hCG (Human chorionic gonadotropin)	Testicular and trophoblastic disease	Blood, urine	[15]
Her-2/neu	Breast	Tissue	[16]
Progesterone receptors	Breast	Tissue	[14]
PSA (Prostate specific antigen)	Prostate	Blood	[17,18]

Table 2.

Examples of immunoassays for tumor markers analysis.

Tumor markers	Biosensor principle	Assay principle	Limit of detection	Refs.
PSA	Fluorescence	Fluorophore-based bio-barcode amplification method	30 nM	[33]
PSA	Microcantilever	Microcantilever	2 nM	[34]
PSA	Electrochemistry	Using gold nanoparticle film electrodes and multienzyme-particle amplification	5 fM	[35]
PSA	Electrochemistry	Carbon nanotube amplification strategies	40 fM	[36]
PSA	Surface-Enhanced Raman Scattering	Immunoassay based on Surface-Enhanced Raman Scattering and immunogold labels	30 fM	[37]
PSA	Colorimetry	Homogenous growth of gold nanocrystals	10 fM	[38]
AFP	Fluorescence	Fluorescence quenching signal of gold nanoparticles	0.17 nM	[39]
AFP	Mass spectrometry	Mass spectrometry signal amplification using small-molecule tagged gold microparticles	1 nM	[40]
AFP	Electrochemistry	Amperometric enzyme immunosensor based on gold nanoparticles and multi-walled carbon nanotube composite membranes	0.6 pM	[41]
AFP	Chemiluminescence	Multilayers enzyme-coated carbon nanotubes as label	0.1 pM	[42]
AFP	Colorimetry	Cascade enzyme-linked immunosorbent assay	0.1 pM	[43]
AFP	Colorimetry	DNAzyme functionalized nano-probes	1.4 pM	[44]
CEA, AFP	Colorimetry	Colorimetric multiplexed immunoassay based on gold nanoparticles	0.02 ng/mL; 0.1 pM	[45]
CEA	Chemiluminescence	Flow-through multianalyte system with substrate zone-resolved technique	0.6 ng/mL	[46]
CEA	Electrochemistry	Layer-by-layer assembly of gold nanoparticles-multi-walled carbon nanotubes-thionine multilayer films	0.01 ng/mL	[47]
CEA	Surface-Enhanced Raman Scattering	Surface-Enhanced Raman Scattering of hollow gold nanospheres	0.01 ng/mL	[48]
CEA	Colorimetry	Enzyme-labeled gold nanoparticle probes	0.012 ng/mL	[49]