In 2002, the McKendry group of IBM ^[1][86] reported a microcantilever array that can sequence-specifically detect unlabeled DNA targets in 80-fold excess of a nonmatching DNA background solution. This proves the excellent anti-interference ability of a genetic-probe-modified microcantilever. In 2003, Su et al. ^[2][121] detected DNA strands by using a microcantilever with gold-nanoparticle-modified genetic probes in dynamic mode. After the amplification process, by catalyzing the nucleation of silver, the method can detect target DNA at a concentration of 0.05 nM or lower. In 2005, Ilic et al. ^[3][122] further improved the dynamic detection sensitivity by using scanning optical–thermomechanical motion excitation method. The sensitivity of their cantilever array was sufficient to detect the binding of a single large biomolecule without labeling. In 2006, Zhang et al. ^[4][123] first demonstrated the nanomechanical analysis of multiple differential gene expression of 1–8U, a potential marker of cancer progression or viral infection, by using cantilever–array sensors in a complex background without amplification or labeling. Also in 2006, Huber et al. ^[5][89] investigated the interaction between dsDNA and two different DNA-binding proteins, the transcription factors SP1 and NF-κB, by using cantilever arrays. This demonstrated the feasibility of micromechanical cantilever sensors for investigating transcription factors. In 2007, Kishan et al. ^[6][124] successful detected small DNA sequences at a femtomolar concentration in human serum by using a 15-mer ssDNA-modified piezoelectrically excited cantilever. In 2010, the group of Miyachi ^[7][125] reported a method of systematic evolution of ligands by an exponential enrichment (SELEX) using a cantilever based on AFM to obtain aptamers that have a strong affinity for target molecules. Thrombin, at concentrations as low as 0.2 nM, can be detected by the AFM-SELEX method ^[8][126]. In 2014, Mishra et al. ^[9][44] modified short nucleic acid sequences onto the microcantilever array surface by using inkjet printing technology. This method improved the detection sensitivity of single-chain peptide nucleic acids (PNA) by about 20-fold, and the detection limit reached the single-base misalignment level. As shown in Figure 18, in 2019, Park et al. ^[10][127] modified microcantilever arrays using DNA probes, Au nanoparticles, and mismatch recognition proteins (MutS). Single-nucleotide polymorphisms (SNPs) of cancer markers can be successfully realized with a detection limit of 100 fM using this method. In addition, to trace detection of nucleic acids and antibodies, a microcantilever modified by genetic probes can also be used for research into DNA enzyme digestion ^[11][128] or DNA strand elasticity ^[12][129].

A virus is an acellular organism that contains only nucleic acid (DNA or RNA), parasitizes cells, and proliferates by replication. Viruses are tiny but harmful, and they have caused global public health problems many times. Therefore, viruses are among the main detection objects of a genetic probe modification microcantilever.

In 2006, Sreepriya et al. ^[13][130] detected feline coronavirus at a concentration of 0.1 μg/mL using a microcantilever modified by feline coronavirus antiserum. The results confirmed the suitability of a genetic-probe-modified microcantilever for the detection of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). In 2007, Hwang et al. ^[14][131] used an RNA aptamer as a microcantilever sensitive layer, and successfully detected hepatitis C virus (HCV) at a concentration of 100 pg/mL. In 2009, Kim et al. ^[15][132] introduced an in/ex situ monitoring method of the HBV by using PZT-embedded microcantilever sensors. The DNA probe (37-mer including T10 spacers) specific to HBV DNA is immobilized on the gold-coated microcantilever to achieve the recognition layer. Moreover, to increase the DNA binding efficiency, an ethylene glycol spacer (HSC11-EG3-OH) is backfilled on the modification layer. In 2010, Cha et al. ^[16][133] reported a dynamic microcantilever biosensor for HBV DNA detection. When using silica nanoparticles (SiNPs) containing rhodamine B isothiocyanate (RITC) for signal amplification, the detection limit of target HBV DNA (243-mer nucleotide) was found to be up to the femtomolar level. In 2012, Shu et al. ^[17][134] successfully detected the grouper nerve necrosis virus using a silicon nitride microcantilever modified by antimicrobial peptides (AMPs). In 2013, Abdullah et al. ^[18][135] successfully detected target ssDNA and ssRNA in human immunodeficiency virus (HIV) by using a silicon-based microcantilever modified by mercapto-oligonucleotides. In 2015, Kim et al. ^[19][136] used the specific primer of human papilloma virus (HPV) as the sensitive layer of silicon-based varistor microcantilever and combined it with PCR amplification to successfully detect HPV. In 2022, Wang et al. ^[20][137] developed an ultrasensitive nanomechanical method based on a microcantilever array and PNA probes for the detection of SARS-CoV-2 virus. The detection process is described in detail in Figure 29, while the method has an extremely low detection limit of 0.1 fM (105 copies/mL) for an N-gene-specific sequence (20 bp). In addition to virus detection, with the help of an AFM system, a microcantilever can also carry out a series of operations such as imaging, operation, and transmission monitoring of a single virus ^[21][138].

The structure of bacteria is much more complicated than that of viruses. For molecular biology testing, specific nucleic acid fragment sequences of pathogenic bacteria are the detection target of a microcantilever.

In 2013, Rijal et al. ^[22][139] detected Escherichia coli O157:H7 (EC) in beef samples by using a piezoelectrically excited cantilever with the toxic gene stx2 as a sensing layer. Compared to the traditional antibody–antigen method (2500 cells/mL), a much lower concentration can be detected by this method without any culture enrichment or amplification (under 700 cells/mL). In 2014, Xu et al. ^[23][140] used porous silica functionalized with NH2 as the medium layer of a piezoresistive microcantilever, then the streptomycin avidin was blocked with bovine serum albumin to form a sensitive layer on the medium layer. The rapid real-time detection of Escherichia coli was successfully realized by using the enzyme cleavage reaction between the sensitive layer and gene stx2 of O157:H7. In 2019, Zheng et al. ^[24][141] fabricated a gold-nanoparticle-amplified microcantilever array biosensor that can determine in parallel ultralow concentrations of foodborne bacteria, including Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Shigella.

In addition to Escherichia coli, in 2015, Khemthongcharoen et al. ^[25][142] detected Vibrio cholerae by combining a gold-coated piezoresistive microcantilever with a self-assembled monolayer (3-mercaptopropionic acid (MPA), for the immobilization of a specific DIVA probe via avidin). As shown in Figure 310, in 2016, Etayash et al. ^[26][143] modified a bimaterial dynamic microcantilever (BMC) with monocyte proliferation monoclonal antibody (mAb) or monocyte proliferation, targeting antibacterial peptide (AMP). With the help of a microfluidic channel integrated on the microcantilever, not only was the detection of Listeria monocytogenes successfully realized, but the response of bacteria to antibiotics could also be monitored in real time. In 2021, Wang et al. ^[27][144] combined an antibody-modified microcantilever with AC thermoelectric technology, which improved the capture efficiency of Vibrio parahaemolyticus and shortened the detection time. Recently, Yersinia ^[28][145] and Mycobacterium tuberculosis ^[29][146] were also successfully detected by a genetic-probe-modified microcantilever sensor. In addition to detection and drug interaction research, a microcantilever can also be used for bacterial growth monitoring ^[30][147].

Cells are the basic structural and functional units of an organism. All organisms except viruses are known to be made up of cells. The structure of cells is complicated, so the targets are specific nucleic acid sequences or another biomarker when detecting cells by a microcantilever.

Cancer cells are one of the highest detection priorities of microcantilever sensors. In 2006, Dell’Atti et al. ^[31][24] immobilized the biotin probe on the surface of a piezoelectric microcantilever through a “glucan–streptomycin” avidin medium layer, and successfully detected mutations in the TP53 gene in leukemia cells. In 2010, Ricciardi et al. ^[32][148] used receptor–ligand and antibody–antigen systems to modify a dynamic microcantilever, and successfully detected the angiogenesis marker Ang-1 of cancer cells. The results also showed that the antibody–antigen method is more advantageous. In 2011, Loo et al. ^[33][73] detected HER2, a biomarker commonly overexpressed in the blood of breast cancer patients, using a magnesium niobate–lead titanate/tin piezoelectric material microcantilever (PEMS). This was the first report of the detection of naturally occurring cancer biomarkers in serum by a cantilever. In 2014, Le et al. ^[34][149] used a silicon nitride microcantilever with a self-assembled monolayer to detect Golgi protein 73, which is a serum biomarker used for diagnosing human hepatocellular carcinoma. The concentration detected was up to 400 ng/mL.

In recent years, with the development of MEMS technology, microcantilevers have been able to detect intact cells. In 2015, Etayash et al. ^[35][150] demonstrated a microcantilever that functionalized with a cancer-specific peptide 18-4 (WxEAAYQrFL) and showed significant deflection on breast cancer cell (MCF7 and MDA-MB-231) binding. The detection limit was 50–100 cells/mL, and the capture yield was 80%. In 2016, Chen et al. ^[36][45] developed a microcantilever array with a TLSIIa aptamer probe for label-free detection of liver cancer cells (HepG2). As shown in Figure 411A, four microcantilevers were modified with aptamers as sensing microcantilevers (pink), and the other four as reference ones (yellow). Δx indicates the differential signal induced by the interaction between aptamers and HepG2 cells. The gray arrow indicates the flow direction of the binding buffer. The detection linear relation ranged from 1 × 10³ to 1 × 10⁵ cells/mL, with a detection limit of 300 cells/mL (S/N = 3).

As shown in Figure 512, in 2018, Etayash et al. ^[37][151], successfully detected breast cancer cells (MDA-MB231) by using a microcantilever array composed of a decapeptide-modified working sensor and a 6-hydroxy-1-hexanethio-modified reference sensor. The research group also used normal mammary epithelial cells (MCF10) as a control group, changing the arrangement of modified probes to study the differences in signal pathways between cancer cells and normal cells. In addition, the microcantilever can be used to study the dynamic deformation difference between cancer cells and normal cells, and the results may indicate a new potential marker to identify cancer cells ^[38][152].

Proteins, which are composed of various amino acid molecules in proportion, are not only an important component of human cells and tissues, but also an important participant in life activities through constant metabolism and renewal in the body.

In 2004, Savran et al. ^[39][153] modified the aptamer probe on the working cantilever surface while there was a nonspecific oligonucleotide probe on the reference cantilever, and successfully recognized specific proteins containing thrombin and Taq DNA polymerase. In 2007, Yoo et al. ^[40][154] modified an avidin-sensitive membrane on the surface of a microcantilever by self-assembly and monitored in real time the binding process of a streptavidin ligand. In 2010, Wang et al. ^[41][155] used a microcantilever modified by a platelet-derived growth factor (PDGF) aptamer probe to quantitatively study the effect of temperature on the binding process between the probe and PDGF. As shown in Figure 613, in their subsequent research from 2013, they integrated a microcantilever array modified by a PDGF aptamer on the microfluidic chip, and built a plug-and-play detection platform with a DVD-ROM as the optical detection module, which can realize fast, low-cost, and parallel detection ^[42][156]. In addition, in 2012, Zhai et al. ^[43][157] modified a microcantilever with RNA aptamers to detect lipid carrier protein (Lipocalin-2). The system showed a detection limit of 4 nM, and the study results also demonstrated that the RNA aptamer can bind to the siderophore binding pocket of the protein. In 2015, Liu et al. ^[44][158] modified a dynamic microcantilever with the biotin–antibiotin system to detect ricin protein. In 2018, the group of Agarwal ^[45][159] detected a heart-type fatty acid-binding protein (h-FABP) in a trace amount (100 ng/mL) by employing a piezoresistive SU-8/CB microcantilever platform for the first time. In 2020, Dilip et al. ^[46][160] tested cardiac troponin-I by using a a SiN–PolySi–SiO₂ composite microcantilever modified with HIgG and Anti-HIgG.

As the most important invention in medical history, antibiotics have become a double-edged sword. The superbacteria produced by the overuse and abuse of antibiotics seriously threaten human health. Therefore, antibiotics are important detection objects of genetic-probe-modified microcantilevers.

In 2008, Ndieyira et al. ^[47][161] successfully used a drug-sensitive mucopeptide analogue (DAla) as a sensitive layer of a microcantilever to detect vancomycin, and quantitatively analyzed the interactions in antibiotic–mucopeptide binding. In 2013, Hou et al. ^[48][162] successfully detected oxytetracycline by using a sensor array consisting of self-assembled monolayers (SAMs) of OTC-specific aptamers as a working cantilever sensitive layer and 6-mercapto-1-hexanol SAMs as a reference cantilever sensitive layer. In the following years, by using a similar method, the group successfully detected multiple antibiotics such as kanamycin ^[49][163], fumonisin B-1 ^[50][164], and nucleolin ^[51][165].

Heavy metals are metals with a density greater than 5 g/cm³. Most heavy metal elements are environmental pollutants, which seriously threaten human health. Therefore, heavy metal ions are also detection targets of microcantilevers. In 2004, Cherian et al. ^[52][166] functionalized a microcantilever with metal-binding protein AgNt84-6 that had the ability to bind multiple ions of Ni²⁺, Zn²⁺, Co²⁺, Cu²⁺, Cd²⁺, and Hg²⁺. This research demonstrated that a microcantilever can be used to discriminate multiple metal ions. In 2009, Xu et al. ^[53][167] grafted a Gly–Gly–His (GGH) tripeptide to the 3-mercaptopropionic acid (MPA) layer on the microcantilever gold surface. Then, they studied the interaction between tripeptide Gly–Gly–His and Cu²⁺ under different environmental conditions and analyzed the mechanism of microcantilever deflection. Since 2012, Peng has functionalized microcantilevers by multiple methods involving sensitive layers for detecting heavy metal ions, including benzo-9-crown-3 doped hydrogen for Be^{2+ [54]}[168], a specific Pb²⁺-dependent DNAzyme molecule for Pb²⁺ ^[55][169], and benzo-9-crown-3 polymer brush for Be^{2+ [56]}[170]. In these studies, the microcantilever sensor modified by DNAzyme not only exhibited high selectivity to Pb²⁺ (10⁻⁸ M), but could be regenerated by flowing through a strong Pb²⁺ chelator (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid). In 2017, You et al. ^[57][171] reported a mass-amplified silver ion sensor (MAIS) based on a resonance microcantilever with cytosine-based DNA as a sensitive layer. To enhance the sensitivity, GNPs that do not affect the specific matching between silver ion and cytosine-DNA layer were introduced to improve resonance frequency shift. Furthermore, genetically modified cantilever sensors can also be used with other environmental pollutants such as okadaic acid (OA) ^[58][172] and in food security, such as with profenofous pesticide residues in vegetables ^[59][173] and the hepatic toxin microcystin–leucine–arginine (MC-LR) ^[60][174].

In early detection, the microcantilever is usually placed in the stage or testing pool with the samples, and then the deflection or vibration changes of the sensor are measured by an instrument such as an AFM or PSD. This detection process not only fails to show the advantages of microcantilever, including small volume, simple structure, and easy integration, but also limits the detection scenario to the laboratory, hindering the application and development of microcantilever sensors in rapid and portable detection. Micro total analysis systems (μTASs), proposed at the beginning of this century, have the characteristics of small size, high integration, and excellent compatibility. Combined with optics, electrochemistry, and other detection methods, the μTAS has become an indispensable analytical technology in the fields of biology, medicine, chemistry, and the environment. Therefore, the μTAS is an ideal choice for a microcantilever to avoid the constraints of the laboratory environment as well as to realize real-time use, miniaturization, and commercialization.

As shown in Figure 714A, in 2006, Lechuga et al. ^[61][67] integrated a silicon-based microcantilever array, polymer microfluidic chip, vertical-cavity surface-emitting laser, segmented photodetector, and other modules to prepare a small biochemical detection system with an Atomic Force Microscopy (AFM) detection limit. As shown in Figure 714B, in 2010, Ricciardi et al. ^[62][175] integrated a microcantilever array and piezoelectric driver into a μTAS chip. With the help of external optical instruments, the chip can complete Salmonella detection within 40 min. As shown in Figure 714C, in 2011, Huang et al. ^[63][64][65][176,177,178] integrated a microcantilever, self-calibrated readout circuit, programmable microcontroller, voltage regulator, wireless transceiver, and other modules on a single-chip SoC by adopting a 0.35 μm standard CMOS process, and then realized wireless detection of HBV by combining it with microfluidic technology. As shown in Figure 714D, in 2015, Khemthongcharoen et al. ^[25][142] prepared an integrated microfluidic detection chip with PCR technology and a pressure-sensitive microcantilever as the core. Compared with conventional PCR, the sensitivity of the integration system is 10 times higher. Starting in 2014, Wang et al., reported a series of works about microcantilever integration, including surface antibody modification ^[66][67][179,180], and the design and fabrication of a microcantilever with cavity, piezoelectric drive, and frequency-tracking circuit ^[68][69][70][181,182,183]. The latest research of this team, in 2021 ^[71][184], had a microcantilever prepared on an SOI substrate by standard CMOS processes such as RIE and PECVD. After modification by the antigen–antibody system, the microcantilever was integrated into a microfluidic chip, as shown in Figure 714E. The integrated detection system can detect alpha-fetoprotein under the amplification of nanoparticles.