J. Zhang

Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3′ and 5′ overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

Label-free protein assay based on a nanomechanical cantilever array

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen–antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml−1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen–antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foreseee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Rapid functionalization of cantilever array sensors by inkjet printing

The controlled deposition of functional layers is the key to converting nanomechanical cantilevers into chemical or biochemical sensors. Here, we introduce inkjet printing as a rapid and general method to coat cantilever arrays efficiently with various sensor layers. Self-assembled monolayers of alkanethiols were deposited on selected Au-coated cantilevers and rendered them sensitive to ion concentrations or pH in liquids. The detection of gene fragments was achieved with cantilever sensors coated with thiol-linked single-stranded DNA oligomers on Au. A selective etch protocol proved the uniformity of the monolayer coatings at a microscopic level. A chemical gas sensor was fabricated by printing thin layers of different polymers from dilute solutions onto cantilevers. The inkjet method is easy to use, faster and more versatile than coating via microcapillaries or the use of pipettes. In addition, it is scalable to large arrays and can coat arbitrary structures in non-contact.

Optimization of DNA Hybridization Efficiency by pH-Driven Nanomechanical Bending

The accessibility and binding affinity of DNA are two key parameters affecting the hybridization efficiency in surface-based biosensor technologies. Better accessibility will result in a higher hybridization efficiency. Often, mixed ssDNA and mercaptohexanol monolayers are used to increase the hybridization efficiency and accessibility of surface-bound oligonucleotides to complementary target DNA. Here, no mercaptohexanol monolayer was used. We demonstrate by differential microcantilever deflection measurements at different pH that the hybridization efficiency peaks between pH 7.5 and 8.5. At low pH 4.5, hydration and electrostatic forces led to tensile surface stress, implying the reduced accessibility of the bound ssDNA probe for hybridization. In contrast, at high pH 8.5, the steric interaction between neighboring ssDNA strands was decreased by higher electrostatic repulsive forces, bending the microcantilever away from the gold surface to provide more space for the target DNA. Cantilever deflection scales with pH-dependent surface hybridization efficiency because of high target DNA accessibility. Hence, by changing the pH, the hybridization efficiency is adjusted.

Development of robust and standardized cantilever sensors based on biotin/NeutrAvidin coupling for antibody detection.

A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA), forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG) and sheep anti-BSA(s-IgG) antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 × 10−3 N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 × 10−3 N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

Nanosensors for cancer detection

Cancer is a major burden in todays society and one of the leading causes of death in industrialised countries. Various avenues for the detection of cancer exist, most of which rely on standard methods, such as histology, ELISA, and PCR. Here we put the focus on nanomechanical biosensors derived from atomic force microscopy cantilevers. The versatility of this novel technology has been demonstrated in different applications and in some ways surpasses current technologies, such as microarray, quartz crystal microbalance and surface plasmon resonance. The technology enables label free biomarker detection without the necessity of target amplification in a total cellular background, such as BRAF mutation analysis in malignant melanoma. A unique application of the cantilever array format is the analysis of conformational dynamics of membrane proteins associated to surface stress changes. Another development is characterisation of exhaled breath which allows assessment of a patients condition in a non-invasive manner.

Chapter 11 - Biological Single Molecule Applications and Advanced Biosensing

1.1. Macroscopic versus microscopic measurements in biology Macroscopic experiments yield time and population averages of the individual characteristics of each molecule. At the level of the individual molecules, the picture is quite different: individual molecules are found in states far from the mean population, and their instantaneous dynamics are seemingly random. Whenever unusual states or the rapid, random motions of a molecule are important, the macroscopic picture fails, and a microscopic description becomes necessary. Single-molecule experiments differ from macroscopic measurements in two fundamental ways: first, in the importance of the fluctuations in both the system and in the measuring instrument, and second, in the relative importance of force and displacement as variables under experimental control and subject to direct experimental measurements. In single-molecule experiments, the crucial parts of the measuring instruments themselves are small and subject to the same fluctuations as the system under study. Single-molecule experiments thus, give access to some of the microscopic dynamics that are hidden in the macroscopic experiments.

Functionalization of Cantilever Array Sensors Using Ink Jet Deposition

Micromechanical cantilever arrays can be used as versatile chemical sensors when coated with a layer that absorbs specific molecules leading to changes of surface stress, mass or temperature [1-4]. After their microfabrication processes, reproducible coating procedures are essential to functionalize the cantilevers individually. Here we introduce the use of ink jet to deposit analyte solutions onto cantilevers for functionalization (Figure 1). The method is fast, reliable and versatile. After evaporation of an Au layer, self-assembled mono-layers of alkanethiols or thiol-linked single-stranded DNA can be formed locally within fractions of a second by spotting droplets of solutions onto selected cantilevers. By selective etch diagnostics, we show down to a microscopic level that the quality of the monolayers is similar to those achieved by microcontact printing [5]. We also spotted thin polymer layers from dilute solutions onto cantilevers. The quality of the coatings and the performance were compared with cantilever sensors coated by previous methods.