High-sensitivity nanosensors for biomarker detection

Abstract

High sensitivity nanosensors utilize optical, mechanical, electrical, and magnetic relaxation properties to push detection limits of biomarkers below previously possible concentrations. The unique properties of nanomaterials and nanotechnology are exploited to design biomarker diagnostics. High-sensitivity recognition is achieved by signal and target amplification along with thorough pre-processing of samples. In this tutorial review, we introduce the type of detection signals read by nanosensors to detect extremely small concentrations of biomarkers and provide distinctive examples of high-sensitivity sensors. The use of such high-sensitivity nanosensors can offer earlier detection of disease than currently available to patients and create significant improvements in clinical outcomes.

Fig. 1

Chemical “nose” sensor. (A) Schematic of the fluorescent polymer interaction with AuNPs. When the polymer interacts with the nanoparticle surface, fluorescence is quenched (left). When the polymer is displaced by a protein target, the fluorescence is restored (right). (B) Protein sensor array made up of chemical noses. A fluorescence pattern is generated based on the specific interaction between the particle and fluorophore. Each well contains different nanoparticle–polymer conjugates. (C) Chemical structure of six different cationic AuNPs that interact with the anionic fluorescent polymer (m ≈ 12) used to sense protein analytes. (D) Fluorescence response patterns of the chemical nose array. (E) Canonical score plot calculated from LDA used to identify seven proteins. Image adapted with permission from ref. .

Fig. 2

Fluorescence activatable nanosensors. (A) Schematic illustration showing the mechanism of flower-like Au–Fe₃O₄ nanoparticles. First the AuNPs and surrounding Fe₃O₄ nanoparticles are synthesized to form a flower like structure. Next, a dye labeled matrix metalloproteinase (MMP) substrate is attached to the probe. When the MMP substrate is intact, the dye is quenched because of the close interaction with the AuNP. When MMP is present, the dye is separated from the AuNP regaining its fluorescence and the probe is activated. (B) In vivo near-IR fluorescence imaging after injection of flower like activatable probes into a mouse model. The probe shows high fluorescence signals at the tumor site where MMP concentrations are high. Modified with permission from ref. .

Fig. 3

In vivo SERS detection of glucose in a mouse model. Schematic image of (A) instrumental set-up for SERS detection of an implantable glucose sensor and (B) Ag film on nanoparticles (AgFON) sensor, showing glucose capture across the surface. (C) Morphology of the sensor as seen by atomic force microscopy. (D) Time course of in vivo glucose measurements. Glucose was infused at 60 minutes (arrow) and measurements were performed by a commercial glucose meter (triangle) and SERS nanosensor (square). Modified with permission from ref. .

Fig. 4

Suspended microchannel resonator. (A) Schematic drawing of a suspended microchannel. This microchannel allows continuous fluid flow through the channel while still achieving sub-femtogram mass resolution by reading the signal from the cantilever under high vacuum. This type of system overcomes the challenge of fluid detection by mechanical nanosensors. Two types of detection by this microchannel is possible: (B) bound molecules by a sandwich assay accumulate in the channel and increase the mass (right) while non-accumulated molecules continue to flow through the channel (left). The frequency therefore shifts due to the change in mass. (C) Non-bound, free-flowing particles within the channel can also by monitored in real-time by peak frequency measurements, as seen in the frequency vs. time graph. (D) Representative frequency shifts over time as antibodies are coated on the suspended microchannel resonator. First a biotin linker is adsorbed on the silicon dioxide surface, then Neutravidin (streptavidin analog) is coated on the channels, and finally biotinylated antibodies bind to the Neutravidin. Between each injection (red bars), rinse cycles were incorporated (blue) where no change in frequency shift is observed. Modified with permission from ref. .

Fig. 5

Electromechanical detection of protein. (A) Representative scanning electron micrographs of trampoline resonators with varying concentrations of prostate specific antigen (PSA) with nanoparticle labels. Scale bar 1 mm. (B) Frequency response based on the PSA concentration showing sensitivity to the fM. Modified with permission from ref. .

Fig. 6

First demonstration of real-time detection of protein using silicon nanowires (SiNW). (A) Schematic figure showing protein binding (right) onto biotin-decorated SiNW (left). (B,C) Conductance versus time graph where the nanowire is originally in buffer solution (region 1) and then (B) 250 nM or (C) 25 pM of streptavidin binds to the nanowire (region 2) and finally the nanowire is in pure buffer (region 3). The arrows indicate where solutions were changed. Modified with permission from ref. .

Fig. 7

Electrical detection using a unique pre-processing method. (A) Primary antibodies to numerous biomarkers are bound to the sensor via a photocleavable crosslinker. (B)Whole blood is injected into the chip (black arrow) and biomarkers bind to the device. (C) The probe is washed and then UV irradiation (orange waves) is applied to cleave the linker between the captured biomarker and sensor. (D) Finally, the antibody–antigen complexes are washed out of the sensor for detection. (E) Response to anti-prostate specific antigen (PSA) purified from a blood sample, initially containing 2.5 ng mL⁻¹ PSA compared with no protein. (F) Normalized response to different concentrations of PSA. Modified with permission from ref. .

Fig. 8

Principle of a magnetic relaxation switches assay using magnetic nanosensors. When monodisperse magnetic nanoparticles conjugated with a binder (i.e. protein, antibody or complementary oligonucleotide sequences), the spin–spin relaxation time (T₂) of neighboring water protons decreased as the self-assembled clusters become more efficient at dephasing nuclear spins of many surrounding water protons. However, when these nanoclusters are treated with a cleaving agent (i.e. enzyme), the nanoparticles become dispersed, switching the T₂ of the solution back to the lower values. These qualities render the developed magnetic nanoparticles as magnetic relaxation switches capable of screening biomarkers by magnetic resonance methods. Modified with permission from ref. .

Fig. 9

Detection of oligonucleotides, proteins, and enzyme activity using magnetic nanosensors. (A) T₂ relaxation time decreased with complementary oligonucleotides when SPIO nanoparticles conjugated with oligonucleotides. (B) T₂ relaxation time decreased with targeted protein-GFP when SPIO nanoparticles conjugated with GFP antibody. (C) T₂ relaxation time increased with the addition of caspase-3 enzyme when SPIO clusters linked with DEVD, a substrate sequence of caspase-3. Modified with permission from ref. .

Fig. 10

Cancer cell detection and profiling using magnetic nanosensors. (A) Magnetic nanosensor bearing carbohydrates was used to profile the carbohydrate-binding characteristics of cancer cells by magnetic resonance imaging. Modified with permission from ref. . (B) Cancer biomarkers detection based on chip-sized microlitre-volume sensors and SPIO-targeting strategies. Modified with permission from ref. .

Fig. 11

The bio-barcode assay technique. (A) The initial probe development of AuNPs. (B) The method of detection for an example protein–prostate specific antigen. Magnetic probes are functionalized with monoclonal antibodies for the protein and mixed with the protein (Step 1). The probes are then separated from the buffer and concentrated on the walls of the tube. The magnetic probes are resuspended in buffer where the secondary probe is introduced. The secondary probe is a AuNP functionalized with polyclonal antibodies and barcode DNA strands. This probe sandwiches the protein target (Step 2). The hybrid particles are separated by magnet again and the barcode DNA is dehybridized (Step 3). The isolated barcode DNA can then be amplified by PCR (Step 4, top) and the probes undergo scanometric DNA detection (Step 5). Modified with permission from ref. .

Scheme 1

Three components necessary for nanosensors.