Method for performing quantitation assays

A method for determining an estimate of a concentration of analyte particles E(C), wherein the analyte particles of a sample of predetermined volume are divided into a number (N) of two or more compartments, at least part of the analyte particles that are present in any of the (N) compartments provide for a measurable signal and the estimated concentration of analyte particles E(C) is a function of the measured signals, characterized in that the method comprises:

• a) determining the number (N) of separate compartments, wherein the number (N) is smaller or equal to the value of the function:
• N
• MAX
• =
• A
• ·
• ln
• ⁡
• (
• C
• +
• C
• -
• )
• ⁢
• /
• ⁢
• σ
• MAX
• 2
• wherein (A) represents a real number equal to 6 or less, preferably 1 or less, more preferably 0.5 and even more preferably 0.2;
• wherein (C+) represents an upper limit of the interval of concentration (C) to be estimated by the method;
• wherein (C−) represents a lower limit of the interval of concentration (C) to be estimated by the method;
• and
• wherein (σMAX) represents a maximum allowable relative standard deviation of the estimate of concentration (C) of analyte particles, wherein C−<C<C+;
• (b) determining a modulation factor (zi), wherein the modulation factor (zi) is a function of volumes (vi) and dilution factors (di) of the sample in at least part of or all of the (N) compartments and partitioning the sample into the (N) compartments, based at least in part on the modulation factor (zi), wherein at least part of or all of the two or more of the (N) compartments comprise or consist of different sample volumes (vi) and/or different dilution factors (di) of the sample, wherein (i) represents an index number of the (N) compartments represented by the integers 0 to N−1, and wherein (vi) represents the volume and (di) represents the dilution factor of the sample in the compartment (i), and wherein the modulation factor (zi) is determined based on a well-defined power sequence, a geometric sequence, an exponential sequence, a polynomial sequence, a distribution in the set of compartments, or a combination thereof, and wherein the set of values of the products (vi) (di) is determined by a function that is non-uniform over a range of variation of the index (i);
• (c) measuring a digital signal from two or more of the (N) compartments and assigning to at least part, preferably all compartments a digital value (ki), wherein compartment (i) is assigned a first value (ki), if the compartment (i) comprises or consists of a predetermined threshold number of analyte particles (mtri) or a predetermined threshold concentration of analyte particles (ctri), preferably one, two, three or more analyte particles in a compartment, and compartment (i) is assigned a second value (ki), if the compartment (i) comprises or consists of less than the threshold number or concentration of analyte particles indicating the first value,
• (d) determining an analog value (τi) for two or more of the (N) compartments, respectively, based on one or more measurements of signals in compartment (i) and based on at least one of:
• a direct measurement of a physical quantity of the sample in the compartment (i);
• a resealing of a direct measurement of a physical quantity of the sample in the compartment (i);
• and
• a mathematical calculation on the basis of a set of direct measurements of the sample in the compartment (i),
• wherein (i) represents the index number of the compartment represented by an integer 0 to N−1, wherein the analog values (τi) depend on the number of analyte particles in the compartment (i) and comprise univocal functions of the number of analyte particles in compartment (i), wherein the values (τi) belong to the ordered set, and wherein at least for one compartment thereof a value (ki) is assigned according to step (c), and
• (e) determining the estimate of concentration of analyte particles E(C) as a function of at least a portion of the values (ki) assigned in step (c) and at least, a portion of the analog values (τi) determined in step (d), whereby the measurement of the digital values (ki) allows the analog valves (τi) to be determinable without external calibration.