Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

Abstract

Rapid progress in identifying disease biomarkers has increased the importance of creating high-performance detection technologies. Over the last decade, the design of many detection platforms has focused on either the nano or micro length scale. Here, we review recent strategies that combine nano- and microscale materials and devices to produce large improvements in detection sensitivity, speed and accuracy, allowing previously undetectable biomarkers to be identified in clinical samples. Microsensors that incorporate nanoscale features can now rapidly detect disease-related nucleic acids expressed in patient samples. New microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes, and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells in the bloodstream more accurately than before. These advances will lead to faster and more reliable clinical diagnostic devices.

Figure 1. Length scales of interest for biomolecular detection

Bioanalytical detection approaches monitor binding events between analytes of interest and specific receptors that occur on the nanoscale. Nanomaterials are ideal for biomolecular display because their dimensions approach the molecular scale, which promotes binding behaviour similar to that observed in solution-based processes. For reference, a molecule of cholesterol is shown on the left; this molecule is nanometre-sized. A DNA molecule (red/blue) and a protein molecule (green/blue), both structures that have sizes on the order of tens of nanometres are also depicted. A bacteriophage (purple), bacterial cell (green) and human cell (pink/blue) span the hundreds of nanometres to micrometre length scale. The microscale length regime is also important for biomolecular detection because most large molecules can only diffuse micrometres while detection is in progress. Devices can straightforwardly be engineered on this length scale, which facilitates the analysis of cells and processing of macroscale clinical samples (depicted as a test tube).

Figure 2. The bio-barcode assay combines micro- and nanoparticles for biomolecular detection

a, A protein target (green diamond) is bound by a magnetic microparticle (grey) functionalized with a specific antibody (green Y-shape). The microparticles are probed with gold nanoparticles (yellow) that are functionalized with antibodies (green) and barcode sequences (blue). After isolation, the barcodes are released and detected on an array. The pattern of barcode binding on the array displaying a variety of barcode-binding sequences reports on the presence or absence of particular analytes. The same approach can be used to detect specific DNA sequences (bottom). b, Data showing HIV p24 Gag protein is detected at picogram levels using the bio-barcode assay described in a. nc, noncomplementary control. Error bars represent standard deviations. c, Clinical data showing sensitivity and specificity of the bio-barcode assay as compared to the gold standard (PCR). The x-axis values correspond to levels of HIV RNA in blood samples drawn from 146 patients measured by PCR. The y axis displays p24 protein concentrations in these same specimens measured by the new barcode assay. Each red dot represents a data point collected from a patient sample, and grey bars indicate the median and interquartile range. This data set shows excellent correspondence between the gold-standard PCR method used to detect HIV RNA and the new barcode assay that measures p24 protein, and there is strong statistical significance of the data for positive versus negative patients. Panels b,c reproduced with permission from ref. , © 2008 Future Medicine Ltd.

Figure 3. Advances in rapid nucleic acid detection based on micro-to-nanoscale engineering of electrode properties

a, A lithographically patterned chip that serves as a substrate for the patterning of nanostructured microelectrodes (NMEs). b, NMEs combine the advantage of a large surface area with a finely nanostructured surface. Gold is electroplated on the surface of the chip through apertures etched in a passivation layer. Probe molecules (blue lines) are attached to the electrodes, and after a target-containing sample (red lines) is introduced, reporter groups are incubated with the sensor to give an electrochemical readout. c, Electron micrographs showing NMEs have 100-μm footprints (top), but feature nanoscale roughness on the order of 10 nm (bottom). d, Nanoscale roughness promotes the creation of a deflection angle that decreases steric interference between probe molecules (blue) immobilized with a thiolated tether (black) on a highly nanostructured surface (orange). The grey ovals represent the zone within which the probe strand has additional space to interact with a target sequence because of the deflection angle. e, Achievement of rapid nucleic acid detection. Data collected when different concentrations of bacterial lysates were incubated with NMEs with 10, 30 and 100 μm footprint for 30 minutes. While all the sensors showed measureable levels of current change (ΔI) when lysates containing a high (150 cells per microlitre; black bars) level of bacteria were introduced, only the largest 100 μm sensors could detect the lower (1.5 cells per microlitre; grey bars) concentration of bacteria, illustrating the importance of the microscale footprint of the sensor. Error bars represent standard error values. f, DNA nanostructures enhance the electrochemical detection sensitivity of nucleic acids. DNA tetrahedral display capture probes with well-defined nanoscale spacing, and target sequences are read-out using an enzymatic label. Figures adapted with permission from: b,d, ref. , © 2011 American Chemical Society; c, ref. , © 2009 American Chemical Society; e,f, ref. , 2012 Nature Publishing Group.

Figure 4. Dividing samples into nano- to femtolitre volumes allows highly accurate quantitation of clinically relevant targets

a, In digital single-molecule amplification, target molecules (blue and red) are compartmentalized into smaller volumes. Nucleic acid amplification of the targets in a much smaller volume than during bulk amplification gives a signal that rises above the background level and above the signal arising from non-specific amplification. The quantitation of positive and negative wells allows a highly precise ratio of two analytes to be determined. This approach provides a wider dynamic range than traditional bulk ensemble measurements when determining the concentrations of target molecules. b, Quantitation of viral RNA within a SlipChip microfluidic device containing wells of different sizes via reverse transcription and PCR. A SlipChip is a piece of plastic or glass patterned with an array of microcompartments that can be filled with sample, and then using a similarly patterned top plate, amplification reagents can be introduced into each well by slipping the position of the two substrates. After the amplification is complete, fluorescent products (green) can be detected visually, and the number of positive wells reports on the concentration of a target analyte. At very low concentrations of RNA, the large wells have a higher probability of capturing and amplifying a molecule of RNA (larger green spots). At high concentrations of RNA, small wells capture and amplify RNA (small green spots). Quantification is performed over the range of concentrations from where several large wells turn green up to where several small wells remain dark. Panel b is reproduced with permission from ref. , © 2011 American Chemical Society.

Figure 5. Microwells separate analytes into femtolitre compartments for quantitative protein detection

a, A target antigen incubated with magnetic beads (blue circle) binds to capture antibodies (orange Y shape) on the bead surface. Biotinylated detection antibodies and an enzymatic reporter (β-galactosidase in this case) form a sandwich complex. b, Once deposited in an array of microwells, a fluorescent substrate is added to the sample, and wells are quantitated to analyse levels of protein biomarkers. c, Analysis of clinical samples for PSA levels. Concentrations of PSA were measured in serum samples from patients who had undergone a radical prostatectomy (blue circles), healthy control samples (red squares) and Bio-Rad PSA control samples (black triangles) determined using a digital ELISA. All samples yielded levels above the detection limit of the digital method (red line). In contrast, the detection limit of a commercially available ELISA test (green line) is too high to provide comparable data. Panel c is reproduced from ref. , 2010 Nature Publishing Group.

Figure 6. Advanced microdevices for circulating tumour cell isolation

a, CTCs (green) are recognized by antibody-functionalized magnetic particles (MNPs; grey). b, A microengineered CTC isolation chip allows negative and positive selection for CTCs by manipulating the flow of microparticle-tagged cells. Whole blood is injected into the device, and passed through an array of microposts that separate the cells based on size. This allows white blood cells (WBCs) and CTCs to be separated from abundant red blood cells (RBCs). Either the WBCs or CTCs can be labelled with magnetic particles, and the application of a magnetic force (B) then forces the different cells to exit the chip via different outlets where they can be isolated as an enriched population. c, CTC levels isolated from blood samples collected from patients with metastatic breast, prostate, pancreatic, colorectal, or lung cancer. The samples were analysed with the iChip or the gold-standard CellSearch method. Significantly higher levels of CTCs were observed when the iChip was used, and many samples that appear negative for CTCs when CellSearch was used for analysis were found to have significant levels of cancer cells with the iChip. This indicates that CTC counting is much more accurate with this new approach and enables CTC monitoring even in patients with low counts. Panels b,c adapted from ref. , © 2013 American Association for the Advancement of Science.