This disclosure provides methods and systems for single-extracellular (EV), multi-omic analysis of target EVs without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target EV from a population of EVs in an encapsulation, derive a plurality of distinct mRNA molecules from the single target EV, and quantify the distinct mRNA molecules to generate an expression profile. Nucleic-acid-tagged antibody conjugates are used for simultaneous proteomic analysis along with the gene expression profiling, which enables classification of an EV in a sample.

A microfluidic system is intended for the analysis of a biological sample containing biological species. The system includes an optical detection device having a source configured to emit an optical signal and at least one sensor having a capture surface defining an optical signal reading zone. The system also includes a microfluidic device having a support in which an amplification chamber, in which an amplification reaction can be carried out, is made, and having an input channel opening into the amplification chamber. The amplification chamber includes at least one first zone located in the sensor reading zone and at least one protuberance forming a recess intended to receive a compound for internal control of the amplification reaction and arranged to be located outside the sensor reading zone or configured to be opaque to said optical signal.

A microfluidic system is intended for the analysis of a biological sample containing biological species. The system includes an optical detection device having a source configured to emit an optical signal and at least one sensor having a capture surface defining an optical signal reading zone. The system also includes a microfluidic device having a support in which an amplification chamber, in which an amplification reaction can be carried out, is made, and having an input channel opening into the amplification chamber. The amplification chamber includes at least one first zone located in the sensor reading zone and at least one protuberance forming a recess intended to receive a compound for internal control of the amplification reaction and arranged to be located outside the sensor reading zone or configured to be opaque to said optical signal.

The present invention relates to a new method for determining the allele frequency and/or mutation rate in nucleic acids, in particular in tumor nucleic acids, in the context of a polymerase chain reaction (PCR), and to diagnostics for this purpose, wherein at least one reference nucleic acid (RN) and one mutation sequence with respect to the reference nucleic acid are used. This reference nucleic acid and mutation sequence allows polymerase chain reaction (PCR) methods to be validated, in particular on the basis of device parameters and sample preparation. Furthermore, the invention relates to an associated diagnosis and prognosis method, in particular for tumor diagnosis as part of a liquid biopsy.

The present invention relates to a new method for determining the allele frequency and/or mutation rate in nucleic acids, in particular in tumor nucleic acids, in the context of a polymerase chain reaction (PCR), and to diagnostics for this purpose, wherein at least one reference nucleic acid (RN) and one mutation sequence with respect to the reference nucleic acid are used. This reference nucleic acid and mutation sequence allows polymerase chain reaction (PCR) methods to be validated, in particular on the basis of device parameters and sample preparation. Furthermore, the invention relates to an associated diagnosis and prognosis method, in particular for tumor diagnosis as part of a liquid biopsy.

A method for determining nitric oxide concentration in biological samples. The method includes for determining nitric oxide concentration in a sample including: (i) providing a nucleic acid complex comprising a first single-stranded nucleic acid molecule comprising a fluorophore crosslinked to the first strand, the fluorophore comprising diaminorhodamine-4-methylamine (DAR-4M) conjugated, to dibenzocyclooctyne-polyethylene glycol (DBCO-PEGn) linker, wherein n equals 4-12 and a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule, wherein the nucleic acid complex further comprises a first label and a targeting moiety conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule, the first label is capable of producing a signal, wherein the intensity of the signal is dependent at least on concentration of the nucleic acid complex in the sample; (ii) contacting the sample with the nucleic acid complex; (iii) measuring the intensity of the signal: and (iii) determining the nitric oxide concentration from the measured signal. Compositions for determining nitric oxide concentrations in biological samples are also included.

A method for determining nitric oxide concentration in biological samples. The method includes for determining nitric oxide concentration in a sample including: (i) providing a nucleic acid complex comprising a first single-stranded nucleic acid molecule comprising a fluorophore crosslinked to the first strand, the fluorophore comprising diaminorhodamine-4-methylamine (DAR-4M) conjugated, to dibenzocyclooctyne-polyethylene glycol (DBCO-PEGn) linker, wherein n equals 4-12 and a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule, wherein the nucleic acid complex further comprises a first label and a targeting moiety conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule, the first label is capable of producing a signal, wherein the intensity of the signal is dependent at least on concentration of the nucleic acid complex in the sample; (ii) contacting the sample with the nucleic acid complex; (iii) measuring the intensity of the signal: and (iii) determining the nitric oxide concentration from the measured signal. Compositions for determining nitric oxide concentrations in biological samples are also included.

A synthetic molecule can be added to a sample at a specified concentration to accurately and/or precisely quantify target molecules included in the sample. The synthetic molecule can include a number of nucleotides. Some of the regions of the synthetic molecule can include sequences that correspond to primers used in an amplification process and other regions of the synthetic molecule can include sequences that are machine-generated. In implementations, an initial number of target molecules included in the sample can be determined based on a number of the target molecules included in an amplification product in relation to the number of synthetic molecules added to the sample.

A synthetic molecule can be added to a sample at a specified concentration to accurately and/or precisely quantify target molecules included in the sample. The synthetic molecule can include a number of nucleotides. Some of the regions of the synthetic molecule can include sequences that correspond to primers used in an amplification process and other regions of the synthetic molecule can include sequences that are machine-generated. In implementations, an initial number of target molecules included in the sample can be determined based on a number of the target molecules included in an amplification product in relation to the number of synthetic molecules added to the sample.

The present disclosure relates to methods of assessing a sample of guide RNAs (gRNAs).