Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets.

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections having diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues.

Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples.

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-µm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 µL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl ß-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000-94,000 unique peptides, corresponding to ∼6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to ∼500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 µg of MCF-7 cell lysate.

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform.

Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ∼1 600 proteins with a median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2 300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.

Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses.

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells.

Extending proteomics to smaller samples can enable the mapping of protein expression across tissues with high spatial resolution and can reveal sub-group heterogeneity. However, despite the continually improving sensitivity of LC-MS instrumentation, in-depth profiling of samples containing low-nanogram amounts of protein has remained challenging due to analyte losses incurred during preparation and analysis. To address this, we recently developed nanodroplet processing in one pot for trace samples (nanoPOTS), a robotic/microfluidic platform that generates ready-to-analyze peptides from cellular material in ~200 nL droplets with greatly reduced sample losses. In combination with ultrasensitive LC-MS, nanoPOTS has enabled >3000 proteins to be confidently identified from as few as 10 cultured human cells and ~700 proteins from single cells. However, the nanoPOTS platform requires a highly skilled operator and a costly in-house-built robotic nanopipetting instrument. In this work, we sought to evaluate the extent to which the benefits of nanodroplet processing could be preserved when upscaling reagent dispensing volumes by a factor of 10 to those addressable by commercial micropipette. We characterized the resulting platform, termed microdroplet processing in one pot for trace samples (µPOTS), for the analysis of as few as ~25 cultured HeLa cells (4 ng total protein) or 50 µm square mouse liver tissue thin sections and found that ~1800 and ~1200 unique proteins were respectively identified with high reproducibility. The reduced equipment requirements should facilitate broad dissemination of nanoproteomics workflows by obviating the need for a capital-intensive custom liquid handling system.

Spatially Resolved Proteome Profiling of <200 Cells from Tomato Fruit Pericarp by Integrating Laser-Capture Microdissection with Nanodroplet Sample Preparation.

Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein expression among constituent cell types. Methods for spatially resolved proteomics are being developed to resolve protein expression for distinct cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser-capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, nanodroplet processing in one pot for trace samples). Tomato fruits were cryosectioned prior to LCM and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ∼0.04 mm2 tissues comprising ∼8-15 parenchyma cells. Spatially resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using ≤200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source-sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.

A fully battery-powered inexpensive spectrophotometric system for high-sensitivity point-of-care analysis on a microfluidic chip.

A cost-effective b[combining low line]a[combining low line]ttery-powered s[combining low line]pectrophotometric s[combining low line]ystem (BASS) was developed for quantitative point-of-care (POC) analysis on a microfluidic chip. By using methylene blue as a model analyte, we first compared the performance of the BASS with a commercial spectrophotometric system, and further applied the BASS for loop-mediated isothermal amplification (LAMP) detection and subsequent quantitative nucleic acid analysis which exhibited a comparable limit of detection to that of Nanodrop. Compared to the commercial spectrophotometric system, our spectrophotometric system is lower-cost, consumes less reagents, and has higher detection sensitivity. Most importantly, it does not rely on external power supplies. All these features make our spectrophotometric system highly suitable for a variety of POC analyses, such as field detection.

Biomarker detection for disease diagnosis using cost-effective microfluidic platforms.

Early and timely detection of disease biomarkers can prevent the spread of infectious diseases, and drastically decrease the death rate of people suffering from different diseases such as cancer and infectious diseases. Because conventional diagnostic methods have limited application in low-resource settings due to the use of bulky and expensive instrumentation, simple and low-cost point-of-care diagnostic devices for timely and early biomarker diagnosis is the need of the hour, especially in rural areas and developing nations. The microfluidics technology possesses remarkable features for simple, low-cost, and rapid disease diagnosis. There have been significant advances in the development of microfluidic platforms for biomarker detection of diseases. This article reviews recent advances in biomarker detection using cost-effective microfluidic devices for disease diagnosis, with the emphasis on infectious disease and cancer diagnosis in low-resource settings. This review first introduces different microfluidic platforms (e.g. polymer and paper-based microfluidics) used for disease diagnosis, with a brief description of their common fabrication techniques. Then, it highlights various detection strategies for disease biomarker detection using microfluidic platforms, including colorimetric, fluorescence, chemiluminescence, electrochemiluminescence (ECL), and electrochemical detection. Finally, it discusses the current limitations of microfluidic devices for disease biomarker detection and future prospects.

A versatile PDMS/paper hybrid microfluidic platform for sensitive infectious disease diagnosis.

Bacterial meningitis is a serious health concern worldwide. Given that meningitis can be fatal and many meningitis cases occurred in high-poverty areas, a simple, low-cost, highly sensitive method is in great need for immediate and early diagnosis of meningitis. Herein, we report a versatile and cost-effective polydimethylsiloxane (PDMS)/paper hybrid microfluidic device integrated with loop-mediated isothermal amplification (LAMP) for the rapid, sensitive, and instrument-free detection of the main meningitis-causing bacteria, Neisseria meningitidis (N. meningitidis). The introduction of paper into the microfluidic device for LAMP reactions enables stable test results over a much longer period of time than a paper-free microfluidic system. This hybrid system also offers versatile functions, by providing not only on-site qualitative diagnostic analysis (i.e., a yes or no answer), but also confirmatory testing and quantitative analysis in laboratory settings. The limit of detection of N. meningitidis is about 3 copies per LAMP zone within 45 min, close to single-bacterium detection sensitivity. In addition, we have achieved simple pathogenic microorganism detection without a laborious sample preparation process and without the use of centrifuges. This low-cost hybrid microfluidic system provides a simple and highly sensitive approach for fast instrument-free diagnosis of N. meningitidis in resource-limited settings. This versatile PDMS/paper microfluidic platform has great potential for the point of care (POC) diagnosis of a wide range of infectious diseases, especially for developing nations.

A microfluidic fully paper-based analytical device integrated with loop-mediated isothermal amplification and nano-biosensors for rapid, sensitive, and specific quantitative detection of infectious diseases.

Bacterial meningitis, an infection of the membranes (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord, is one of the major causes of death and disability worldwide. Higher case-fatality rates and short survival times have been reported in developing countries. Hence, a quick, straightforward, and low-cost approach is in great demand for the diagnosis of meningitis. In this research, a microfluidic fully paper-based analytical device (µFPAD) integrated with loop-mediated isothermal amplification (LAMP) and ssDNA-functionalized graphene oxide (GO) nano-biosensors was developed for the first time for a simple, rapid, low-cost, and quantitative detection of the main meningitis-causing bacteria, Neisseria meningitidis (N. meningitidis). The results can be successfully read within 1 hour with the limit of detection (LOD) of 6 DNA copies per detection zone. This paper device also offers versatile functions by providing a qualitative diagnostic analysis (i.e., a yes or no answer), confirmatory testing, and quantitative analysis. These features make the presented µFPAD capable of a simple, highly sensitive, and specific diagnosis of N. meningitis. Furthermore, this microfluidic approach has great potential in the rapid detection of a wide variety of different other pathogens in low-resource settings.

Recent innovations in cost-effective polymer and paper hybrid microfluidic devices.

Hybrid microfluidic systems that are composed of multiple different types of substrates have been recognized as a versatile and superior platform, which can draw benefits from different substrates while avoiding their limitations. This review article introduces the recent innovations of different types of low-cost hybrid microfluidic devices, particularly focusing on cost-effective polymer- and paper-based hybrid microfluidic devices. In this article, the fabrication of these hybrid microfluidic devices is briefly described and summarized. We then highlight various hybrid microfluidic systems, including polydimethylsiloxane (PDMS)-based, thermoplastic-based, paper/polymer hybrid systems, as well as other emerging hybrid systems (such as thread-based). The special benefits of using these hybrid systems have been summarized accordingly. A broad range of biological and biomedical applications using these hybrid microfluidic devices are discussed in detail, including nucleic acid analysis, protein analysis, cellular analysis, 3D cell culture, organ-on-a-chip, and tissue engineering. The perspective trends of hybrid microfluidic systems involving the improvement of fabrication techniques and broader applications are also discussed at the end of the review.

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-ß-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for 'all-in-one' one-pot sample preparation. This 'all-in-one' method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

Rapid and Accurate Diagnosis of the Respiratory Disease Pertussis on a Point-of-Care Biochip.

BACKGROUND: Pertussis is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis (B. pertussis). The infection is difficult to diagnose especially in underserved or resource-limited areas. We developed a low-cost and instrument-free diagnostic method for rapid and accurate detection of B. pertussis on a point-of-care (POC) testing device. METHODS: We developed a paper/polymer hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) method for the rapid and accurate detection of B. pertussis. This microfluidic approach was validated by testing 100 de-identified remnant clinical nasopharyngeal swabs and aspirates, which were confirmed to be either positive or negative for B. pertussis by a validated real-time PCR assay at the Children's Hospital Los Angeles. FINDINGS: The instrument-free detection results could be successfully read by the naked eye within 45 min with a limit of detection (LOD) of 5 DNA copies per well. Our optimized bacterial lysis protocol allowed the direct testing of clinical samples without any complicated sample processing/preparation (i.e. DNA extraction) or the use of any equipment (e.g. centrifuges). The validation of the microfluidic approach was accomplished by testing 100 clinical samples. High sensitivity (100%) and specificity (96%) with respect to real-time PCR were achieved. INTERPRETATION: This microfluidic biochip shows great potential for point-of-care disease diagnosis in various venues including schools and physician's offices, especially in low-resource settings in developing nations. FUNDING: NIH/NIAID under award number R21AI107415, NIH RCMI Pilot Grant, the Philadelphia Foundation, the Medical Center of the Americas Foundation.

A low-cost microfluidic platform for rapid and instrument-free detection of whooping cough.

Whooping cough also called Pertussis is a highly contagious respiratory infection that affects all age populations. Given recent pertussis outbreaks, there is an urgent need for a point-of-care (POC) device for rapid diagnosis of pertussis. Herein, we report a low-cost microfluidic POC device integrated with loop-mediated isothermal amplification (LAMP) technique for the rapid and accurate diagnosis of pertussis. The 3D-printed bioanalyzer housed not only the biochip but also an in-house-developed portable and fully battery-powered heater for rapid POC detection of pertussis, without the need of external electricity. The fluorescence-based results could be rapidly visualized in about one hour by the naked eye without the need for any additional instrumentation. In addition, a simple centrifuge-free sample preparation process was optimized for the efficient lysis of pertussis samples and successfully used for direct detection of bacteria in nasopharyngeal samples. High sensitivity, with a limit of detection (LOD) of 5 DNA copies per LAMP zone, and high specificity were demonstrated. We envision that the microfluidic POC device can be used in various venues such as medical clinics, schools, and other low-resource settings for the fast detection of pertussis.

Recent advances of controlled drug delivery using microfluidic platforms.

Conventional systematically-administered drugs distribute evenly throughout the body, get degraded and excreted rapidly while crossing many biological barriers, leaving minimum amounts of the drugs at pathological sites. Controlled drug delivery aims to deliver drugs to the target sites at desired rates and time, thus enhancing the drug efficacy, pharmacokinetics, and bioavailability while maintaining minimal side effects. Due to a number of unique advantages of the recent microfluidic lab-on-a-chip technology, microfluidic lab-on-a-chip has provided unprecedented opportunities for controlled drug delivery. Drugs can be efficiently delivered to the target sites at desired rates in a well-controlled manner by microfluidic platforms via integration, implantation, localization, automation, and precise control of various microdevice parameters. These features accordingly make reproducible, on-demand, and tunable drug delivery become feasible. On-demand self-tuning dynamic drug delivery systems have shown great potential for personalized drug delivery. This review presents an overview of recent advances in controlled drug delivery using microfluidic platforms. The review first briefly introduces microfabrication techniques of microfluidic platforms, followed by detailed descriptions of numerous microfluidic drug delivery systems that have significantly advanced the field of controlled drug delivery. Those microfluidic systems can be separated into four major categories, namely drug carrier-free micro-reservoir-based drug delivery systems, highly integrated carrier-free microfluidic lab-on-a-chip systems, drug carrier-integrated microfluidic systems, and microneedles. Microneedles can be further categorized into five different types, i.e. solid, porous, hollow, coated, and biodegradable microneedles, for controlled transdermal drug delivery. At the end, we discuss current limitations and future prospects of microfluidic platforms for controlled drug delivery.

Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells.

Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separations for in-depth proteome profiling of low-nanogram samples. Peptides are first separated using high-pH LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of HeLa digest. Furthermore, in combination with a recently developed nanowell-based sample preparation workflow, we demonstrated deep proteome profiling of >6000 protein groups from small populations of cells, including ∼650 HeLa cells and 10 single human pancreatic islet thin sections (∼1000 cells) from a pre-symptomatic type 1 diabetic donor.

A paper/polymer hybrid CD-like microfluidic SpinChip integrated with DNA-functionalized graphene oxide nanosensors for multiplex qLAMP detection.

A paper/poly(methyl methacrylate) (PMMA) hybrid CD-like microfluidic SpinChip integrated with DNA probe-functionalized graphene oxide (GO) nanosensors was developed for multiplex quantitative LAMP detection (mqLAMP). This approach can simply and effectively address a major challenging problem of multiplexing in current LAMP methods.

Multiplexed instrument-free meningitis diagnosis on a polymer/paper hybrid microfluidic biochip.

Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae), and Haemophilus influenzae type b (Hib) are three most common pathogens accounting for most bacterial meningitis, a serious global infectious disease with high fatality, especially in developing nations. Because the treatment and antibiotics differ among each type, the identification of the exact bacteria causing the disease is vital. Herein, we report a polymer/paper hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) for multiplexed instrument-free diagnosis of these three major types of bacterial meningitis, with high sensitivity and specificity. Results can be visually observed by the naked eye or imaged by a smartphone camera under a portable UV light source. Without using any specialized laboratory instrument, the limits of detection of a few DNA copies per LAMP zone for N. meningitidis, S. pneumoniae and Hib were achieved within 1h. In addition, these three types of microorganisms spiked in artificial cerebrospinal fluid (ACSF) were directly detected simultaneously, avoiding cumbersome sample preparation procedures in conventional methods. Compared with the paper-free non-hybrid microfluidic biochip over a period of three months, the hybrid microfluidic biochip was found to have a much longer shelf life. Hence, this rapid, instrument-free and highly sensitive microfluidic approach has great potential for point-of-care (POC) diagnosis of multiple infectious diseases simultaneously, especially in resource-limited settings.

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers.

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings.

Nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer.

A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays.