Nanoparticles for Proteins and Cells Detection

Novel Tools for Clinical Diagnostics

  • Fig. 1: Immunoassays using gold nanoparticles as: (up) red-ox labels on magnetic beads platforms; (down) carriers of enzymatic labels in an ELISA format. Adapted from [5] and [6].Fig. 1: Immunoassays using gold nanoparticles as: (up) red-ox labels on magnetic beads platforms; (down) carriers of enzymatic labels in an ELISA format. Adapted from [5] and [6].
  • Fig. 1: Immunoassays using gold nanoparticles as: (up) red-ox labels on magnetic beads platforms; (down) carriers of enzymatic labels in an ELISA format. Adapted from [5] and [6].
  • Fig. 2: Proteins detection using nanoparticles. (Up) Electrochemical set-up and scheme of an immunoassay for protein detection in human serum, using gold nanoparticles as catalytic labels on magnetic beads platforms.; (down) SEM images of the gold nanoparticles (small points) attached to the magnetic beads (big spheres). Adapted from [7].
  • Fig. 3: Cancer cells detection using gold nanoparticles. (Up) Tumor cells cultured on the electrotransducers; (down) scheme of a tumor cell sensor based of gold nanoparticle labels and electrocatalytic detection. Adapted from [8].
  • Fig. 4: Scheme of the protein sensing system based on nanochannels immunoblocking for the detection of a cancer biomarker in whole human blood, as well as SEM images of the nanoporous membranes. Adapted from [10].

Proteins and cells detection methodologies with interest for rapid and costeffective diagnostics and based on nanoparticles and nanochannels, are described. The developed devices are based on the use of screen-printing technology, a mass production technology, with interest even to fulfill market needs. The described simple nanomaterial-based devices are being offered as alternative to sophisticated and high-cost diagnostic equipment that requires experts for their use.

Nanoparticles and Electrochemical Biosensors

Biosensing technology is advancing rapidly and taking advantage of the latest developments in materials science and in particular from the nanomaterials (NM) field. Because of the electrical and electrochemical qualities of NMs, interesting developments in the design of novel bioassays are being reported. Between various NMs, nanoparticle (NP) based biosensing is offered as excellent screening alternative to existing conventional strategies/assays with interest for clinical analysis, environmental monitoring as well as safety and security [1,2].

NPs label based electrochemical biosensors offer several advantages in terms of cost-efficiency in comparison to traditional methods of bioanalysis such as ELISA or PCR [3]. Noble metals based NPs, especially gold nanoparticles (AuNPs) are advantageous due to their simple synthesis, narrow size distribution, unique properties (structural, electronic, magnetic, optical, and catalytic) and easy bioconjugation possibilities.

Different strategies for the electrochemical detection of NPs used as labels in immunosensing and cell sensing are presented. The optimized biosensors have been applied for the detection of biomolecules of clinical interest, such as tumor cells or cancer biomarkers in a biological fluid (i.e. blood).

Nanoparticles as Redox Labels

Metallic NPs, such as AuNPs have redox properties that allow their direct detection without their previous dissolution. In the case of the AuNPs (20 nm sized) the methodology consists in the adsorption of the NPs onto the electrode surface followed by the application of an oxidative potential in HCl.

The nanoparticle is oxidized generating tetrachloroaurate ions which are then reduced back to gold metal while applying a differential pulse voltammetric (DPV) scan, which in turn gives a peak of current as analytical signal.

The size effect of the AuNPs (ranging between 5–80 nm) on their voltammetric response following the above detailed method on screen-printed carbon electrotransducers (SPCEs) has also been recently studied, finding that the electrochemical properties of AuNP suspensions are strongly depended on the size and the hydrodynamic properties of the solvent [4].

The advantages of the AuNP direct voltammetric detection strategy have allowed its application for the detection of biomolecules in different bioassays formats. Magnetosandwich immunoassays for the detection of human IgG as model protein have been performed, using paramagnetic microbeads (2.8 μm sized) as platforms and AuNPs as labels, achieving a detection limit of 260 pg ml-1 [5].

The use of these magnetic platforms allows samples preconcentration and minimizing of matrix effects. AuNPs also can act as carriers of other labels. For example, the signaling antibodies conjugated with AuNPs can also be previously modified with an enzymatic label (i.e. HRP), so the NP can act as carrier of a high number of HRP and exerting an amplification effect on i.e. optical enzyme-linked immunosorbent assays (ELISA).

This effect has been approached for the detection of CA15-3, an important biomarker present in blood samples and useful for the follow-up of the medical treatment of breast cancer, achieving a better sensitivity and shorter assay time in comparison to classical ELISA procedures without NPs [6] (fig. 1). The same carrying effect of AuNPs can be applied in electrochemical sensors explained before.

Nanoparticles as Catalytic Labels

An electrocatalytic strategy applied for the detection of proteins consists in using the AuNPs to enhance the formation of H2 (Hydrogen Evolution Reaction, HER). The presence of AuNPs on the electrode surface shifts the reduction potential of the hydrogen ions. By applying an adequate electrodeposition potential a catalytic current used as signal is registered.

This detection route combined with magnetosandwich immunoassay formats has been applied for the detection of anti-hepatitis B virus antibodies (up to 3 mUI ml-1) in human serum samples [7] (fig. 2). This sensing system is more rapid, cheaper and easier to be handled in comparison to conventional techniques. Electrocatalytic method based on HER has also been applied for detection of cancer cells. This sensing is based on the direct incubation of the cells onto the surface of the SPCE, followed by their recognition through the interaction between the cell surface proteins and specific antibodies labeled with AuNPs.

The final detection of the AuNPs through the HER allows to detect 4.000 cells per 700 μl of sample, even in the presence of control/interfering cells [8] (fig. 3). Although the detection limit is high the method can still be improved and probably be with interest for cancer cells detection.

Nanochannels and Immunoblocking Using Nanoparticles

Nanopores, obtained in both synthetic and biological membranes, have been extensively used as resistive-pulse sensors for molecular and macromolecular analytes. The most reported applications of nanopores/nanochannels for electrochemical analysis are mainly focused on DNA sensing, but also protein sensing has been achieved. However, in most of these biosensing devices the experimental setup is hard to be built. The measurement is tedious and time-consuming and the achieved sensitivity is low.

In this context alternatives are needed to bring this technology to real sample biosensing applications. The capability of current tuning of a nanopore/ nanochannel-based platform upon immunoblocking by taking advantage of an electrotransducer fabricated by screen-printing technology and a simple voltammetric detection mode has been shown as an easy and rapid alternative that overcome the above mentioned drawbacks.

This nanochannel based biosensing has been firstly used for the rapid and simple label-free electrochemical detection of proteins using SPCEs modified with nanoporous alumina membranes. The membranes have a high pore density and small pore diameter, which result in a substrate with high surface area that can be easily functionalized with antibodies through covalent binding.

After that, the immunorecognition event with the specific antigen takes place, giving rise to the pore blocking. The blockage inside the nanochannels is fast, pore size dependent and easy to be detected by measuring the decrease in the DPV peak current of the Fe2+/3+ indicator [9]. Furthermore, the efficiency of the nanochannels to act not only as sensing platforms but also as filters of complex matrixes was approached for the detection of a cancer biomarker spiked in whole human blood samples.

Constituents of the blood such as the cells (on the micrometric scale) remain on the surface of the membrane which acts as a filter, while the cancer biomarker can enter through the channels and be captured by the specific antibody previously immobilized. This system was applied for the detection at clinical relevant levels ( up to 52 U ml-1) of CA15-3 breast cancer biomarker. In this case, the sensitivity of the labelfree assay was highly improved using AuNP tags as blocking agents in sandwich immunoassays [10] (fig. 4).

Conclusions and Future Challenges

Nanoparticles (NPs) represent the most interesting nanomaterials for the design of novel electrochemical systems for proteins and cells sensing. Their use in biosensing technologies is mostly related to labeling approaches. To obtain the biosensing signal NPs detection can be achieved according to various direct or indirect (i.e. catalytic properties or blocking abilities being inside nanochannels) electrochemical detection methodologies.

Due to their rich chemistry the use of NPs as carriers of other electroactive or optical active labels is shown to be of great interest too. The developed NP based electrochemical biosensing systems are more rapid, cheaper and easier to be performed in comparison to the traditional techniques which use other labels (i.e. dyes or enzymes). In addition these techniques may be expected to be robust enough so as to be offered as alternative to the conventional ones for real point of care applications with interest for diagnostics, environmental monitoring, safety and security beside other industries.

Although the progress reported in NP based biosensing systems there are still various issues that need to be better addressed before this technology could come to the market. A better integration of the entire assay that would lead to compact, flexible and cost efficient- devices beside reproducibility and stability issues need still to be addressed. This is why this interesting and promising research field is still progressing and trying to solve more challenges.

Acknowledgements

We acknowledge MICINN (Madrid) for the projects PIB2010JP-00278 and IT2009-0092, the E.U.’s support under FP7 contract number 246513 “NADINE’’ and the NATO Science for Peace and Security Programme’s support under the project SfP 983807.

References

[1] De la Escosura-Muñiz A. et al.: Materials Today 12 (7-8), 24–34 (2010)

[2] De la Escosura-Muñiz A. et al.: Expert Opin. Med. Diagn. 4(1), 21–37 (2010)

[3] De la Escosura-Muñiz A. et al.: TrAC 27, 568–584 (2008).

[4] De la Escosura-Muñiz A. et al.: Nanoscale 3, 3350–3356 (2011)

[5] Ambrosi A. et al.: Anal. Chem. 79 (14), 5232–5240 (2007)

[6] Ambrosi A. et al.: Anal. Chem. 82 (3), 1151–1156 (2010)

[7] De la Escosura-Muñiz et al.: Biosen. Bioelectron. 26, 1710–1714 (2010)

[8] De la Escosura-Muñiz et al.: Anal. Chem. 81(24), 10268–10274 (2009)

[9] De la Escosura-Muñiz et al.: Electrochem. Commun. 12, 1501–1504 (2010)

[10] De la Escosura-Muñiz et al.: Small 7(5), 675–682 (2011)

Contact

Prof. Arben Merkoçi

Institut Català de Nanotecnologia, Campus UAB,

08193 Bellaterra, Barcelona, Spain

arben.merkoci@icn.cat

www.icn.cat www.nanobiosensors.org

Authors

Register now!

The latest information directly via newsletter.

To prevent automated spam submissions leave this field empty.